Method for sorting particles

ABSTRACT

A method for sorting a particle of interest from a plurality of particles includes the steps of determining an absorption maxima of the particle of interest, providing a light source for generating a beam of coherent light at a wavelength correlating to the absorption maxima, providing a plurality of particles on a support surface, and imparting relative motion between the beam of coherent light and the plurality of particles so as to cause differential movement between the particle of interest and the plurality of particles. The particle of interest is then collected.

RELATED APPLICATIONS

This application is a continuation-in-part of Application Serial No.09/845,245, filed Apr. 27, 2001, entitled “Methods and Apparatus for Useof Optical Forces for Identification, Characterization and/or Sorting ofParticles”.

This application is related to Application Serial No. 09/843,902, filedon Apr. 27, 2001, entitled “Method for Separating Micro-Particl s”, asamended, with named inventor Osman Kibar, which claims priority fromprovisional Application Serial No. 60/248,451, entitled “Method andApparatus for Sorting Cells or Particles”, filed Nov. 13, 2000. Thoseapplications are incorporated herein by reference as if fully set forthherein.

FIELD OF THE INVENTION

This invention relates to methods and apparatus for the selection,identification, characterization, and/or sorting of materials utilizingat least optical or photonic forces. More particularly, the inventionsfind utility in biological systems, generally considered to be the useof optical forces for interaction with bioparticles having an opticaldielectric constant.

BACKGROUND OF THE INVENTION

Separation and characterization of particles has a wide variety ofapplications ranging from industrial applications, to biologicalapplications, to environmental applications. For example, in the fieldof biology, the separation of cells has numerous applications inmedicine and biotechnology. Historically, sorting technologies focusedon gross physical characteristics, such as particle size or density, orto utilize some affinity interaction, such as receptor-ligandinteractions or reactions with immunologic targets.

Electromagnetic response properties of materials have been utilized forparticle sorting and characterization. For example, dielectrophoreticseparators utilize non-uniform DC or AC electric fields for separationof particles. See, e.g., U.S. Pat. No. 5,814,200, Pethig et al.,entitled “Apparatus for Separating By Dielectrophoresis”. Theapplication of dielectrophoresis to cell sorting has been attempted. InBecker (with Gascoyne) et al., PNAS USA, Vol. 92, pp. 860-864, January1995, Cell Biology, in the article entitled “Separation of Human BreastCancer Cells from Blood by Differential Dielectric Affinity”, theauthors reported that the dielectric properties of diseased cellsdiffered sufficiently to enable separation of the cancer cells fromnormal blood cells. The system balanced hydrodynamic anddielectrophoretic forces acting on cells within a dielectric affinitycolumn containing a microelectrode array. More sophisticated separationsystems have been implemented. See, e.g., Cheng, et al., U.S. Pat. No.6,071,394, “Channel-Less Separation of Bioparticles on a BioelectronicChip by Dielectrophoresis”. Yet others have attempted to useelectrostatic forces for separation of particles. See, e.g., Judy etal., U.S. Pat. No. 4,440,638, entitled “Surface Field-Effect Device forManipulation of Charged Species”, and Washizu “ElectrostaticManipulation of Biological Objects”, Journal of Electrostatics, Vol. 25,No. 1, June 1990, pp. 109-103.

Light has been used to sort and trap particles. One of the earliestworkers in the field was Arthur Ashkin at Bell Laboratories, who used alaser for manipulating transparent, μm-size latex beads. Ashkin's U.S.Pat. No. 3,808,550 entitled “Apparatuses for Trapping and AcceleratingNeutral Particles” disclosed systems for trapping or containingparticles through radiation pressure. Lasers generating coherent opticalradiation were the preferred source of optical pressure. The use ofoptical radiation to trap small particles grew within the Ashkin BellLabs group to the point that ultimately the Nobel Prize was awarded toresearchers from that lab, including Steven Chu. See, e.g., Chu, S.,“Laser Trapping of Neutral Particles”, Sci. Am., p. 71 (February 1992),Chu, S., “Laser Manipulation of Atoms and Particles”, Science 253, pp.861-866 (1991).

Generally, the interaction of a focused beam of light with dielectricparticles or matter falls into the broad categories of a gradient forceand a scattering force. The gradient force tends to pull materials withhigher relative dielectric constants toward the areas of highestintensity in the focused beam of light. The scattering force is theresult of momentum transfer from the beam of light to the material, andis generally in the same direction as the beam. The use of light to trapparticles is also sometimes referred to as an optical tweezerarrangement. Generally, utilizing the Rayleigh approximation, the forceof trapping is given by the following equation:$F_{g} = {2{\pi \cdot r^{3}}\frac{\sqrt{ɛ_{B}}}{c}\left( \frac{ɛ - ɛ_{B}}{ɛ + {2ɛ_{B}}} \right)\left( {\nabla{\cdot I}} \right)}$

where F_(g) is the optical gradient force on the particle in thedirection toward the higher intensity, r is the radius of the particle,∈_(B) is the dielectric constant of the background medium, ∈ is thedielectric constant of the particle, I is the light intensity in wattsper square centimeter and ∇ is the spatial derivative. FIG. 1 shows adrawing of a particle in an optical tweezer. The optical tweezerconsists of a highly focused beam directed to the particle.

As shown in FIG. 1, the focused beam 12 first converges on the particle10 and then diverges. The intensity pattern 14 relates to thecross-section of the intensity of the beam in the horizontal dimension,and the intensity pattern 16 is the cross-section of intensity in thevertical dimension. As can be seen from the equation, the trapping forceis a function of the gradient of the intensity of the light. Thus, theforce is greater where the light intensity changes most rapidly, andcontrarily, is at a minimum where the light intensity is uniform.

Early stable optical traps levitated particles with a vertical laserbeam, balancing the upward scattering force against the downwardgravitational force. The gradient force of the light served to keep theparticle on the optical axis. See, e.g., Ashkin, “Optical Levitation byRadiation Pressure”, Appl. Phys. Lett., 19(6), pp. 283-285 (1971). In1986, Ashkin disclosed a trap based upon a highly focused laser beam, asopposed to light propagating along an axis. The highly focused beamresults in a small point in space having an extremely high intensity.The extreme focusing causes a large gradient force to pull thedielectric particle toward that point. Under certain conditions, thegradient force overcomes the scattering force, which would otherwisepush the particle in the direction of the light out of the focal point.Typically, to realize such a high level of focusing, the laser beam isdirected through a high numerical aperture microscope objective. Thisarrangement serves to enhance the relative contribution from the highnumerical aperture illumination but decreases the effect of thescattering force.

In 1987, Ashkin reported an experimental demonstration of opticaltrapping and manipulation of biological materials with a single beamgradient force optical trap system. Ashkin, et al., “Optical Trappingand Manipulation of Viruses and Bacteria”, Science, 20 March, 1987, Vol.235, No. 4795, pp. 1517-1520. In U.S. Pat. No. 4,893,886, Ashkin et al.,entitled “Non-Destructive Optical Trap for Biological Particles andMethod of Doing Same”, reported successful trapping of biologicalparticles in a single beam gradient force optical trap utilizing aninfrared light source. The use of an infrared laser emitting coherentlight in substantially infrared range of wavelengths, there stated to be0.8 μm to 1.8 μm, was said to permit the biological materials to exhibitnormal motility in continued reproductivity even after trapping forseveral life cycles in a laser power of 160 mW. The term “opticution”has become known in the art to refer to optic radiation killingbiological materials.

The use of light to investigate biological materials has been utilizedby a number of researchers. Internal cell manipulation in plant cellshas been demonstrated. Ashkin, et al., PNAS USA, Vol. 86, 7914-7918(1989). See also, the summary article by Ashkin, A., “Optical Trappingand Manipulation of Neutral Particles Using Lasers”, PNAS USA, Vol. 94,pp. 4853-4860, May 1997, Physics. Various mechanical and forcemeasurements have been made including the measurement of torsionalcompliance of bacterial flagella by twisting a bacterium about atethered flagellum. Block, S., et al., Nature (London), 338, pp. 514-518(1989). Micromanipulation of particles has been demonstrated. Forexample, the use of optical tweezers in combination with a microbeamtechnique of pulsed laser cutting, sometimes also referred to as laserscissors or scalpel, for cutting moving cells and organelles wasdemonstrated. Seeger, et al., Cytometry, 12, pp. 497-504 (1991). Opticaltweezers and scissors have been used in all-optical in vitrofertilization. Tadir, Y., Human Reproduction, 6, pp. 1011-1016 (1991).Various techniques have included the use of “handles” wherein astructure is attached to a biological material to aid in the trapping.See, e.g., Block, Nature (London), 348, pp. 348-352 (1990).

Various measurements have been made of biological systems utilizingoptical trapping and interferometric position monitoring withsubnanometer resolution. Svoboda, Nature (London), 365, pp. 721-727(1993). Yet others have proposed feedback based systems in which atweezer trap is utilized. Molloy, et al., Biophys. J., 68, pp. 2985-3055(1995).

A number of workers have sought to distort or stretch biologicalmaterials. Ashkin in Nature (London), 330 pp. 769-771 (1987), utilizedoptical tweezers to distort the shape of red blood cells. Multipleoptical tweezers have been utilized to form an assay to measure theshape recovery time of red blood cells. Bronkhorst, Biophys. J., 69, pp.1666-1673 (1995). Kas, et al., has proposed an “optical stretcher” inU.S. Pat. No. 6,067,859 which suggests the use of a tunable laser totrap and deform cells between two counter-propagating beams generated bya laser. The system is utilized to detect single malignant cancer cells.Yet another assay proposed colliding two cells or particles undercontrolled conditions, termed the OPTCOL for optical collision. See,e.g., Mammer, Chem & Biol., 3, pp. 757,763 (1996).

Yet others have proposed utilizing optical forces to measure a propertyof an object. See, e.g., Guanming, Lai et al., “Determination of SpringConstant of Laser-Trapped Particle by Self-Mining Interferometry”, Proc.of SPIE, 3921, pp. 197-204 (2000). Yet others have utilized the opticaltrapping force balanced against a fluidic drag force as a method tocalibrate the force of an optical trap. These systems utilize the highdegree of dependence on the drag force, particularly Stokes drag force.

Yet others have utilized light intensity patterns for positioningmaterials. In U.S. Pat. No. 5,245,466, Burnes et al., entitled “OpticalMatter”, arrays of extended crystalline and non-crystalline structuresare created using light beams coupled to microscopic polarizable matter.The polarizable matter adopts the pattern of an applied, patterned lightintensity distribution. See also, “Matter Rides on Ripples of Lights”,reporting on the Bums work in New Scientist, Nov. 18, 1989, No. 1691.Yet others have proposed methods for depositing atoms on a substrateutilizing a standing wave optical pattern. The system may be utilized toproduce an array of structures by translating the standing wave pattern.See, Celotta et al., U.S. Pat. No. 5,360,764, entitled “Method ofFabricating Laser Controlled Nanolithography”.

Yet others have attempted to cause motion of particles by utilizinglight. With a technique termed by its authors as “photophoresis”, BrianSpace, et al., utilized a polarized beam to induce rotary motion inmolecules to induce translation of the molecules, the desired goal beingto form a concentration gradient of the molecules. The techniquepreferably utilizes propeller shaped molecules, such that the inducedrotary motion of the molecules results in translation.

Various attempts have been made to form microfluidic systems, put tovarious purposes, such as sample preparation and sorting applications.See, e.g., Ramsey, U.S. Pat. No. 6,033,546, entitled “Apparatus andMethod for Performing Microfluidic Manipulations for Chemical Analysisand Synthesis”. Numerous companies, such as Aclara and Caliper, areattempting to form micro-systems comprising a ‘lab on a chip’.

Others have attempted to combine microfabricated devices with opticalsystems. In “A Microfabricated Device for Sizing and Sorting DNAMolecules”, Chou, et al., PNAS USA, Vol. 96, pp. 11-13, January 1999,Applied Physical Sciences, Biophysics, a microfabricated device isdescribed for sizing and sorting microscopic objects based upon ameasurement of fluorescent properties. The paper describes a system fordetermining the length of DNA by measuring the fluorescent properties,including the amount of intercalated fluorescent dye within the DNA. In“A Microfabricated Fluorescence-Activated Cells Sorter”, NatureBiotechnology, Vol. 17, November 1999, pp. 1109-1111, a “T”microfabricated structure was used for cell sorting. The system utilizeda detection window upstream of the “T” intersection and based upon thedetected property, would sort particles within the system. A forwardsorting system switched fluid flow based upon a detected event. In areverse sorting mode, the fluid flow was set to route all particles to awaste collection, but upon detection of a collectible event, reversedthe fluid flow until the particle was detected a second time, afterwhich the particle was collected. Certain of these systems are describedin Quake et al., PCT Publication WO 99/61888, entitled “MicrofabricatedCell Sorter”.

Yet others have attempted to characterize biological systems based uponmeasuring various properties, including electromagnetic radiationrelated properties. Various efforts to explore dielectric properties ofmaterials, especially biological materials, in the microwave range havebeen made. See, e.g., Larson et al., U.S. Pat. No. 4,247,815, entitled“Method and Apparatus for Physiologic Facsimile Imaging of BiologicTargets Based on Complex Permittivity Measurements Using RemoteMicrowave Interrogation”, and PCT Publication WO 99/39190, namedinventor Hefti, entitled “Method and Apparatus for Detecting MolecularBinding Events”.

Despite the substantial effort made in the art, no comprehensive,effective, sensitive and reliable system has been achieved.

SUMMARY OF THE INVENTION

The methods and apparatus of this relate generally to the use of lightenergy to obtain information from, or to apply forces to, particles. Theparticles may be of any form which have a dielectric constant. The useof light for these beneficial purposes is the field of optophoresis. Aparticle, such as a cell, will have a Optophoretic constant or signaturewhich is indicative of a state, or permits the selection, sorting,characterization or unique interaction with the particle. In thebiological regime, the particles may include cells, organelles,proteins, or any component down to the atomic level. The techniques alsoapply in the non-biological realm, including when applied to allinorganic matter, metals, semiconductors, insulators, polymers and otherinorganic matter.

Considering the biological realm, the cell represents the true point ofintegration for all genomic information. Accessing and deciphering thisinformation is important to the diagnosis and treatment of disease.Existing technologies cannot efficiently and comprehensively address theenormous complexity of this information. By unlocking the fundamentalproperties of the cell itself, the methods and apparatus describedherein create new parameters for cellular characterization, cellularanalysis and cell-based assays.

This technology represents a practical approach to probing the innerworkings of a particle, such as a living cell, preferably without anydyes, labels or other markers. The “Optophoretic Constant” of a celluniquely reflects the physiological state of the cell at the exactmoment in which it is being analyzed, and permits investigation of theinner workings of cells. These techniques allow simple and efficientgathering of a wide spectrum of information, from screening new drugs,to studying the expression of novel genes, to creating new diagnosticproducts, and even to monitoring cancer patients. This technologypermits the simultaneous analysis and isolation of specific cells basedon this unique optophoretic parameter. Stated otherwise, this technologyis capable of simultaneously analyzing and isolating specific particles,e.g. cells, based on their differences at the atomic level. Used aloneor in combination with modern molecular techniques, the technologyprovides a useful way to link the intricate mechanisms involving theliving cell's overall activity with uniquely identifiable parameters.

In one aspect, the invention is a method for the characterization of aparticle by the steps of observing a first physical position of aparticle, optically illuminating the particle to subject it to anoptical force, observing the second physical position of the particle,and characterizing the particle based at least in part upon reaction ofthe particle to the optical force. The characterization may be that theparticle, e.g., a cell, has a certain disease state based upon thedetected optophoretic constant or signature.

While characterization may be done with or without physical separationof multiple particles, a method for separating particles may consist of,first, subjecting particles to optical gradient force, second, movingthe particle, and third, separating desired particle from otherparticles. The particle may be separate from the others by furtheroptical forces, by fluidic forces, by electromagnetic forces or anyother force sufficient to cause the required separation. Separation mayinclude segregation and sorting of particles.

In yet another aspect, the invention includes a method for analyzingparticles by electrokinetically moving the particles, and subjecting theparticles to optical forces for sorting. The electrokinetic forces mayinclude, for example, eletroosmosis, electrophoresis anddielectrophoresis.

In addition to the use of the dielectric aspects of the particle forcharacterization and sorting, certain of the inventive methods may beused to determine the dielectric constant of a particle. One methodconsists of subjecting the particle to an optical gradient force in aplurality of media having different dielectric constants, monitoring themotion of the particle when subject to the optical gradient force in thevarious media, and determining the dielectric constant of the particlebased upon the relative amount of motion in the various media.

Yet other methods permit the sorting of particles according to theirsize. One method includes the steps of subjecting the particles to aoptical fringe pattern, moving the fringes relative to the particles,wherein the improvement comprises selecting the period of the fringes tohave a differential effect on differently sized particles. An alliedmethod sorts or otherwise separates particles based upon the particlesflexibility when subject to a optical force. One set of exemplary stepsincludes: subjecting the particles to an optical pattern having fringes,the fringe spacing being less than the size of the particle in anuncompressed state, moving the fringes relative to the medium containingthe particles, and whereby particles having relatively higherflexibility are separated from those with relatively lower flexibility.

In addition to the use of optical gradient forces, the systems andmethods may use, either alone or in combination with other forces, theoptical scattering force. One method for separation in an optophoresisset up consists of providing one or more particles, subjecting theparticles to light so as to cause a scattering force on the particles,and separating the particles based upon the reaction to at least thescattering force.

Various techniques are described for enhancing the sensitivity anddiscrimination of the system. For example, a sensitive arrangement maybe provided by separating the particles in a medium having a dielectricconstant chosen to enhance the sensitivity of the discrimination betweenthe particles, and changing the medium to one having a dielectricconstant which causes faster separation between the particles. Oneoption for enhancing the sensitivity is to choose the dielectricconstant of the medium to be close to the dielectric constant of theparticles.

Accordingly, it is an object of this invention to provide a method ofidentification, characterization, selection and/or sorting of materialshaving an optical dielectric constant.

It is yet a further object of this invention to provide a system forsorting or identifying particles without labeling or otherwise modifyingthe particle.

It is yet another object of this invention to provide a system in whichuncharged or neutral particles may be sorted or otherwise characterized.

Yet another object of this invention is to provide a system in whichparticles may be manipulated remotely, thereby reducing thecontamination to the system under study.

It is yet another object of this invention to provide a system forcharacterizing, moving and/or sorting particles that may be used inconjunction with other forces, without interference between the opticalforces and the other forces.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical depiction of optical intensity patterns for aprior art optical tweezer system, showing both the focus beam, aparticle and the cross-section of intensity of the beam.

FIG. 2 is a cross-sectional drawing of the optical system forinterfering two beams utilizing a variable path length by moving amirror.

FIG. 3 is a schematic diagram of a system utilizing interference betweentwo beams where the path length is varied utilizing a phase modulator.

FIG. 4 is a cross-sectional drawing of an optical system utilizing aninterferometer where the path length is adjustable via a phasemodulator, and FIG. 4A is a side view of an alternate opticalarrangement utilizing counterpropagating beams for particle levitation.

FIG. 5 is a cross-sectional drawing of an optical system including aninterferometer and a phase modulator for changing the optical pathlength, and includes a photograph of a wave pattern generated by thesystem.

FIG. 6 is a cross-sectional drawing of an optical system utilizingseparate illumination and imaging systems.

FIG. 7 is a depiction of an optical system interfacing with a fluidicsystem.

FIG. 8 is a cross-sectional drawing of an optical system utilizing amoving scanning system.

FIGS. 9A and 9B are cross-sectional drawings of an optical systemincluding a mask based generation of intensity pattern.

FIG. 10 is a side view of an array of illumination sources, illuminatinga substrate or support.

FIGS. 11A, 11B and 11C show graphs of intensity, forces and potentialenergy, respectively, as a function of position in one exemplaryembodiment of the invention.

FIG. 12A shows two particles at first positions and a superimposedoptical pattern.

FIG. 12B shows the particles at second positions after illumination bythe optical pattern.

FIG. 12C shows the trapping of particle B in an optical trap.

FIGS. 13A, 13B and 13C show graphs of the potential energy as a functionof distance for the technique for separating particles.

FIGS. 14A and 14B show graphical depictions of particle sorting from aone-dimensional particle source, in FIG. 14A showing the particle flowand in FIG. 14B showing particles transported in a fluid flow.

FIG. 15 is a plan view drawing of a “T” channel sorting structure.

FIG. 16 is a plan view of an “H” sorting structure.

FIG. 17 is a plan view of a “Y” shaped sorting structure.

FIG. 18 is a plan view of a “X” channel sorting structure.

FIG. 19 is a perspective view of a two-dimensional sorting structure.

FIG. 20 is a plan view of a multi-dimensional sorting structure.

FIG. 21 is a side view of a multi-dimensional sorting structureincluding a reflective surface for generation of the optical gradientpattern.

FIG. 22 is a side view of a sorting structure including a capturestructure.

FIG. 23 is a plan view of a microfluidic system including a recyclepath.

FIG. 24 is a plan view of a particle analysis system utilizing particledeformability as a factor in the selection or characterization.

FIG. 25 is a plan view of a sorting or characterization system utilizingthe particle size relative to the optical gradient periodicity as afactor.

FIG. 26 is a system for separation of particles utilizing the scatteringforce of light for separation.

FIG. 27A is a perspective drawing of a scattering force switch.

FIG. 27B is a plan, side view of a scattering force switch.

FIG. 27C is a plan, side view of a scattering force switch with the beamon.

FIG. 28 is a schematic drawing of a system for determining thedielectric constant of particles in various fluidic media of varyingdielectric constant.

FIG. 29 is a cross-sectional drawing of particles and a light intensityprofile for separating particles in a dielectric medium.

FIG. 30 is a perspective view of a optical tweezer array.

FIG. 31 is a graph of molar extinction coefficient as a function ofwavelength for hemoglobin-O₂ absorption spectrum.

FIG. 32 shows time lapse photographs of an experiment separatingparticles by size with a moving optical gradient field.

FIG. 33 shows time lapse photographs of an experiment separatingparticles by surface functionalization.

FIG. 34 shows a Before, After and Difference photograph of particlessubject to a moving optical gradient field.

FIG. 35 is a graph of percent of cells measured in an experiment versusescape velocity, for a variety of cell types.

FIG. 36 shows photographs of sorting of two cell types in a microchanneldevice. 1 shows a red blood cell and a white blood cell successivelyentering the moving optical gradient field. 2 shows that white bloodcell has been translated down by the action of the moving opticalgradient field while the red blood cell has escaped translation. 3 and 4show that the red blood cell and white blood cell continue to flow intoseparate channels, completing the sorting.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following definitions are provided for an understanding of theinvention disclosed herein.

“Dielectric constant” is defined to be that property which determinesthe electrostatic energy stored per unit volume for unit potentialgradient. (See, e.g., the New IEEE Standard Dictionary Of Electrical AndElectronics Terms, ©1993).

The “optical dielectric constant” is the dielectric constant of aparticle or thing at optical wavelengths. Generally, the opticalwavelength range is from 150 Å to 30,000 Å.

An “optical gradient field” is an optical pattern having a variation inone or more parameters including intensity, wavelength or frequency,phase, polarization or other parameters relating to the optical energy.When generated by an interferometer, an optical gradient field orpattern may also be called an optical fringe field or fringe pattern, orvariants thereof.

A “moving optical gradient field” is an optical gradient field thatmoves in space and/or time relative to other components of the system,e.g., particles or objects to be identified, characterized, selectedand/or sorted, the medium, typically a fluidic medium, in contact withthe particles, and/or any containment or support structure.

An “optical scattering force” is that force applied to a particle orthing caused by a momentum transfer from photons to material irradiatedwith optical energy.

An “optical gradient force” is one which causes a particle or object tobe subject to a force based upon a difference in dielectric constantbetween the particle and the medium in which it is located.

“Optophoresis” or “Optophoretic” generally relates to the use ofphotonic or light energy to obtain information about or spatially moveor otherwise usefully interact with a particle.

“Optophoretic constant” or “optophoretic signature” or “optophoreticfingerprint” refer to the parameter or parameters which distinguish orcharacterize particles for optical selection, identification,characterization or sorting.

An “optical tweezer” is a light based system having a highly focusedbeam to a point in space of sufficiently high intensity that thegradient force tends to pull a dielectric particle toward the point ofhighest intensity, typically with the gradient force being sufficientlystrong to overcome the scattering force. Most typically, the laser beamis directed through a microscope objective with a high numericalaperture, with the beam having a diffraction limited spot size ofapproximately the wavelength of the light, 5,000 to 20,000 Å, thoughmore typically 10,000 Å. Generally, an optical tweezer has a beam widthin the focal plane of 2 μm or less, and typically about 1 μm.

“Separation” of two objects is the relative spatial distancing over timeof a particle from some other reference point or thing.

“Sorting” involves the separation of two or more particles in ameaningful way.

DESCRIPTION OF EXEMPLARY APPARATUS

Optical Components—Generation of Moving Optical Gradient Field

FIGS. 2-10 describe various systems for generation of optical patterns,sometimes termed fringe patterns or optical fringe patterns, including,but not limited to, a moving optical gradient field pattern. Theseexemplary embodiments are intended to be illustrative, and not limiting,as other apparatus may be utilized to generate the optical fields andforces to achieve the desirable results of these inventions.

The points raised in discussions of specific embodiments may beconsidered to be generally applicable to descriptions of the otherembodiments, even if not expressly stated to be applicable.

The light source for use with systems has certain generally desirableproperties. As to wavelength, the wavelength will generally be chosenbased upon one or more considerations. In certain applications, it maybe desirable to avoid damage to biological materials, such as cells. Bychoosing wavelengths in ranges where the absorption by cellularcomponents, mostly water, are minimized, the deleterious effects ofheating may be minimized. Wavelengths in the range from approximately0.3 μm to approximately 1.8 μm, and more preferably, from substantially0.8 to substantially 1.8 μm, aid in reducing biological damage. However,even for biological applications, a laser having a wavelength generallyconsidered to be damaging to biological materials may be used, such aswhere the illumination is for a short period of time where deleteriousabsorption of energy does not occur. In yet other applications, it maybe desirable to choose a wavelength based upon a property of theparticle or object under consideration. For example, it may be desirableto choose the wavelength to be at or near an absorption band in order toincrease (or decrease) the force applied against a particle having aparticular attribute. Yet another consideration for wavelength choicemay be compatibility with existing technology, or a wavelength naturallygenerated by a source. One example would be the choice of the wavelengthat 1.55 μm. Numerous devices in the 1.55 μm wavelength region existcommercially and are used extensively for telecommunicationsapplications.

Generally, the light sources will be coherent light sources. Mosttypically, the coherent light source will consist of a laser. However,non-coherent sources may be utilized, provided the system can generatethe forces required to achieve the desired results. Various laser modesmay be utilized, such as the Laguerre-Gaussian mode of the laser.Furthermore, if there is more than one light source in the system, thesesources can be coherent or incoherent with respect to each other.

The spot size or periodicity of the intensity pattern is preferablychosen to optimize the effective results of the illumination. In manyapplications, it is desirable to have a substantially uniform gradientover the particle, e.g., cell, to be interrogated such that thedielectric properties of the entire particle (cell) contribute to theresulting force. Broadly, the range varies from substantially 1 tosubstantially 8 times the size (diameter or average size) of theparticle or object, more preferably, the range is from substantially 2to substantially 4 times the size. Various methods and systems known tothose skilled in the art may be utilized to achieve the desired spotsize or periodicity, e.g., using a defocused beam or a collimated beamhaving the desired size. The typical characterization of the radius ofthe spot is the 1/e² radius of the beam intensity. For manyapplications, including cellular applications, the beam size will be onthe order of 10 microns, though sometimes as small as five microns, andin even certain other occasions, as small as two microns. In certainapplications, it is desirable to have the periodicity of theillumination in the range from substantially 1 to substantially 2 timesthe size (diameter or average size) of the particle or object. For manybiological applications, a periodicity of from substantially 5 μm to 25μm, and more preferably from 10 μm to 20 μm. Certain applications mayutilize smaller sizes, e.g., for bacteria, or larger sizes, e.g., forlarger particles. In yet other applications, it may be desired toutilize a spot size smaller than the particle or object, such as whereinterrogation of a sub-cellular region is desired.

The examples of systems for generating intensity patterns, describedbelow, as well as other systems for generating intensity patterns usefulfor the subject inventions include various optical components, as wellas a control system to generate the desired pattern, intensity profileor other gradient, such as a moving optical field gradient. Variousoptical systems may be adapted for use in the systems of the invention,so as to effectively carry out the methods and achieve the resultsdescribed herein. Exemplary systems which may be adapted in whole or inpart include: Young's slits, Michelson interferometer, Mach-Zenderinterferometer, Haidinger circular fringe systems, Fresnel mirrorinterferometer, plane-parallel plate interferometer, Fabry-Perotinterferometer and any other system for generating an optical gradientintensity pattern or fringe pattern.

Turning now to a detailed description of exemplary systems for use withthe subject inventions. FIG. 2 shows an optical component description ofa system 20 generally configured to generate a moving optical gradientfield pattern to provide a force on one or more particles provided tothe system 20. The optical forces may then be used for characterization,identification, selection and/or sorting of the particles. A lightsource 22, preferably a laser, generates a first beam 24 directed towardbeam splitter 26. Beam splitter 26 may be of any mode or type known tothe art, such as a prism beam splitter, consistent with the goals andobjects of this invention. A first transmitted beam 28 passes throughthe beam splitter 26. A first reflected beam 30 reflects from the beamsplitter 26 to a reflective surface 32, typically a mirror, to generatea second reflected beam 34. The first transmitted beam 28 and secondreflected beam 34 interfere and generate an intensity pattern 38,generally being located at the operative portion of the slide or support36 where the light would interact with the particle or object ofinterest. The optical pattern 38 moves relative to other objects, e.g.,the particles, the substrate, and/or the fluidic medium containing theparticles, by virtue of a change in the optical path length between thefirst transmitted beam 28 and the combination of the first reflectedbeam 30 and second reflected beam 34. Mirror 32 is movable, by actuator40. One example of an actuator 40 could comprise a motor and screwsystem to move mirror 32. Numerous alternative structures for movingmirror 32 are known to the art, e.g., piezoelectric systems, oscillatingmirror systems and the like.

FIG. 3 shows a two-beam interference based system. A source of coherentlight, such as laser 52, generates a first beam 54 directed to a beamsplitter 56. A first reflected beam 58 is directed toward the sampleplate 70 and a first transmitted beam 60 is directed to a modulator,such as a phase modulator 62. The phase modulator 62 may be of any typeknown to those skilled in the art. Phase modulator 62 is under controlof the control system 64 and results in modulated beam output 66 whichis directed to a mirror 74. The modulated beam 66 reflects from mirror74 to generate the second reflected beam 68 which is directed to thesample plate 70. The first reflected beam 54 and second reflected beam68 generate a pattern 72 at the operative interface with the sampleplate 70. The control system 64 is connected to the phase modulator 62so as to cause the pattern 72 to move relative to the objects within thesystem 50, such as the sample plate 70.

FIG. 4 shows an optical component diagram of an interferometer system80. A light source, such as laser 82, generates a first light beam 84directed to beam splitter 86. An interferometer composed of the firstmirror 88 and second mirror 90 generate an output beam 100 having thedesired beam properties, including the desired gradient properties. Thefirst beam 84 passes through beam splitter 86 to generate a firsttransmitted beam 94 directed to first mirror 88. The reflected beamretraces path 94 to the beam splitter 86. The first reflected beam 96passes through phase modulator 92 to generate first modulated beam 98directed to the second mirror 90. The reflected beam from second mirror90 retraces the path 98 through the phase modulator 92 and beam 96 tothe beam splitter 86. The beam 100 is output from the interferometersection of the system 80 and directed toward the microscope objective104.

The objective 104 is directed toward the sample plate 106. Optionally, amirror 108, most preferably a planar mirror, may be disposed beneath thesample plate 106. The mirror 108 is oriented so as to provide reflectedlight onto the sample plate 106 bearing or containing the particles orobjects under analysis or action of the system 80. The scattering forcecaused by the beam 102 as initially illuminates the sample plate 106 maybe counteracted, in whole or in part, by directing the reflectedradiation from mirror 108 back toward the sample. As discussed more inthe section relating to surface effects, below, the reflected light andthe upward scattering force reduce the overall effects of the scatteringforces, such that the gradient forces may be more effectively utilized.

FIG. 4 includes an optional imaging system. The light 102 from theobjective 104 is reflected by the beam splitter 120 generating thirdreflected beam 110 which is directed toward imaging optics 112. Theoptics 112 image the light on a detector 114, such as a charge coupledevice (CCD) detector. The output of the detector 114 may be provided toan imaging system 116. The imaging system 116 may optionally include adisplay, such as a monitor (CRT, flat panel display, plasma display,liquid crystal display, or other displays known to those skilled in theart). The imaging system 116 may optionally include image enhancementsoftware and image analysis software, recording capability (to tape, tooptical memory, or to any other form of memory known to those skilled inthe art).

A control system 118 controls the modulator 92 so as to generate thedesired optical force pattern within the system 80. Optionally, theimaging system 116 may be coupled to the control system 118. A feedbacksystem may be created whereby the action of the particles on the sampleplate 106 may be imaged through the system 116 and then utilized in thecontrol system analysis to control the operation of the overall system80.

FIG. 5 shows a interferometer based system 120. A light source, such aslaser 122, generates a first beam 124 directed toward an optionalspatial filter 126. The spatial filter 126 would typically includelenses 128 and a spatial filter aperture 130. The aperture typically isround. The spatial filters serves to collimate the laser beam and toproduce a smooth intensity profile across the wavefront of the laserbeam. The interferometer 140 includes first mirror 146 and second mirror144, as well a beam splitter 142. The phase modulator 148 is disposedwithin one of the two arms of the interferometer 140.

As shown in FIG. 5, a mirror 132 is optionally disposed to reflect thelight from the source 122 to the interferometer 140. As will beappreciated by those skilled in the art, optical systems may include anynumber or manner of components designed to transfer or direct lightthroughout the system. One such example is the planar mirror 132 whichmerely serves to direct the radiation from one major component, e.g.,the spatial filter, to another major component, e.g., the interferometer140. In addition to mirrors, other common transfer components mayinclude fiber optics, lenses, beam splitters, diffusers, prisms,filters, and shaped mirrors.

Beam 150 exits the interferometer 140 and is directed toward objective152 and imaged at or near the sample plate 154. As shown, a dichroicmirror 170 serves to reflect the light 150, but to also permit passageof light from source 168, such as a fiber providing radiation from asource through the dichroic mirror 170 and objective 152 to illuminatethe operative regions of the sample plate 154.

Optionally, a detection system may be disposed to image the operativeportions of the sample plate 154. As shown, objective 156 is disposedbeneath the sample plate 154, with the output radiation beingtransferred via mirror 158 to an imaging apparatus 164, such as a chargecouple device (CCD). Optionally, an infrared filter 160 may be disposedwithin the optical path in order to select the desired wavelengths fordetection. The output of the detector 164 is provided to an imagingsystem 166. As described in connection with other figures, the imagingsystem 166 may include image enhancement and image analysis software andprovide various modes of display to be user. Optionally, the imagingsystem 166 is coupled to the control system 172 such as when used forfeedback.

FIG. 6 shows an optical system having illumination of a sample plate 194from the top side and imaging from the bottom side. A laser 180generates a first beam 182 which optionally passes through a spatialfilter 184. The spatial filter as shown includes lens 184 and aperture188. The output of the spatial filter 184 passes through the objective192 and is imaged onto the sample plate 194. The sample plate 194 andmaterial supported on it may be imaged via an objective 196. An optionalmirror 198 directs radiation to an optional filter 200 through animaging lens 202 onto the detector 204. The detector 204 is coupled toan imaging system 206. Preferably, the imaging system 206 providesinformation to a control system 208 which controls various opticalcomponents of the system.

FIG. 7 shows an optical system interfacing a sample plate which includesbounded structures. The system 210 includes a sample plate 212 whichoptionally includes microfluidic channels. Alternatively, the sampleplate 212 may support a separate structure containing the microfluidicchannels. As one exemplary structure formed from the microfluidicchannels, a “T” sorting arrangement is shown for a simple, thoughuseful, example. An input reservoir 216 connects to a first channel 218which terminates in a T at intersection 220. A first output channel 222couples to a first output reservoir 224. A second output channel 226couples to a second output chamber 228. As shown, the input chamber iscoupled to ground and the first output chamber 224 and second outputchamber 228 are connected to −V. The fluidic channel structures arediscussed in more detail, below.

The microscope objective 232 serves to both provide the opticalradiation to the sample plate 222 as well as to provide the imaging ofthe system. A light source 238, such as a laser, or more particularly, alaser diode, generates light which may be imaged by optics 240. Adichroic beam splitter 236 directs the radiation to the microscopeobjective 232. As shown, the objective has a magnification power of 100.For the biological applications, a magnification range of from 1 to 200is desired, and more preferably, from 10 to 100. The objective 232 has a1.25 numerical aperture. The preferable range of numerical apertures forthe lenses is from 0.1 to 1.50, and more preferably from 0.4 to 1.25.The output from the objective 232 passes through the beam splitter 236,reflects from optional mirror 242 through optics (e.g., lens) 244,through the optional filter 246 to the imaging device 280. The imagingdevice, shown as a CCD, is connected to the imaging system 282. Theoutput of the imaging system 282 is optionally coupled to the controlsystem 284. As shown, the control system 284 controls both thetranslation stage 232 connected to the sample plate 212, as well as tothe light source 238.

FIG. 8 shows a system for generating an intensity pattern within thescanned area 260. An input beam 262, such as from a coherent lightsource, such as a laser, is directed toward the system. A firstoscillating component 264, such as a galvanometer or resonant scanner,intercepts the input beam 262 and provides a first degree of motion tothe beam. The beam is directed to a polygonal mirror 268 which containsmultiple faces 270. As the polygonal mirror 268 rotates around axis 272,the light is swept across the scanner area 260. Lens 274 are provided asrequired to appropriately image the light into the scanned area 260.Optionally, a mask or other pattern 276 may be disposed within theoptical pathway so as to provide for the variation of the optical forceswithin the scanned area 260. Any of a wide variety of techniques forgenerating either the oscillatory motion or the scanning via thepolygonal mirror are known to those skilled in the art.

FIG. 9 shows a system utilizing masks to generate an optical forcepattern. A source 280, such as a laser, generates a beam 282 directed totoward a mask 284. Optionally, a phase modulator 290 may be disposedbetween the source 280 and the mask 284. Optionally, the mask 284 may bemoved, such as by actuator 286, which may be a motor, piezoelectricdriven system, microelectromechanical (MEMs), or other drivingstructures known to those skilled in the art. The optical mask 284creates a desired light intensity pattern adjacent the sample plate 288.The optical mask 284 may modulate any or all of the components of thelight passing there through, include, but not limited to, intensity,phase and polarization. The mask 284 may be a holographic mask which, ifused, may not necessarily require coherent light. Other forms of masks,such as spatial light modulators may be utilized to generate variationsin optical parameters.

Yet another mirror arrangement consists of utilizing a micromirrorarrangement. One such micromirror structure consists of an array ofmirrors, such as utilized in the Texas Instrument Digital Micromirrorproduct.

FIG. 10 shows an alternate system for illumination in which multiplesources 290 are directed toward the sample plate or surface 294. Eachsource 290 is controlled by control system 296, with the various outputs292 from the sources 290 illuminating the surface of the support 294.

Arrays of sources 290 may be fabricated in many ways. One preferablestructure is a vertical cavity surface emitting laser (VCSEL) array.VCSEL arrays are known to those skilled in the art and serve to generateoptical patterns with control of the various lasers comprising theVCSELs. Similarly, laser diode bars provide an array of sources.Alternatively, separate light sources may be coupled, such as throughfiber optic coupling, to a region directed toward the surface 294.

The imaging system may serve function beyond the mirror imaging of thesystem. In addition to monitoring the intensity, size and shape of theoptical fringes, it may be used for purposes such as calibration.

Optical Forces

The apparatus and methods of the instant inventions utilize, at least inpart, forces on particles caused by light. In certain embodiments, alight pattern is moved relative to another physical structure, theparticle or object, the medium containing the particle or object and/orthe structure supporting the particle or object and the medium. Oftentimes, a moving optical pattern, such as moving optical gradient fieldmoves relative to the particles. By moving the light relative toparticles, typically through a medium having some degree of viscosity,particles are separated or otherwise characterized based at least inpart upon the optical force asserted against the particle. While most ofthe description describes the light moving relative to other structures,it will be appreciated that the relative motion may be achievedotherwise, such as by holding the light pattern stationary and movingthe subject particle, medium and/or support structure relative to theoptical pattern.

FIGS. 11A, 11B and 11C depict, respectively, the optical intensityprofile, the corresponding optical force on a particle or cell and thecorresponding potential energy of the particle in the optical intensityprofile as a function of distance (x). FIG. 11A shows the intensityprofile generated and applied against one or more particles. As shown,the intensity varies in a undulating or oscillating manner. Theintensity, as shown, shows a uniform periodicity and symmetric waves.However, the intensity variations may be symmetric or asymmetric, or ofany desired shape. The period may be fixed or may be variable. FIG. 11Bshows the absolute value of the force as a function of position. Theforce is the spatial derivative of the intensity. FIG. 11C shows thepotential energy as a function of position. The potential energy is theintegrated force through a distance.

The profiles of FIGS. 11A-11C are shown to be generally sinusoidal.Generally, such a pattern would result from interference fringes.Differing profiles (of intensity, force and potential energy) may bedesired. For example, it may be desirable to have a system where thepotential energy well is relatively flat at the bottom and has steepersides, or is asymmetric in its form.

FIGS. 12A and 12B show two particles, labeled “A” and “B”. in FIG. 12A,the particles are shown being illuminated by a two-dimensional intensitypattern 300. FIG. 12B shows the position of particles A and B at a latermoment of time, after the intensity pattern has moved to position 302.In this example, the optical force has caused particle B to moverelative to its prior position. Since the effect of the optical pattern300 on particle A was less than on particle B, the relative positions ofparticles A and B are different in FIG. 12B as compared to FIG. 12A.

In one implementation of the system, the position of particles A and Bin FIG. 12A would be determined. The system would then be illuminatedwith the desired gradient field, preferably a moving optical gradientfield, and the system then imaged at a later point in time, such asshown in FIG. 12B. The absence of motion, or the presence of motion(amount of motion, direction of motion, speed of motion, etc.) may beutilized to characterize, or analyze the particle or particles. Incertain applications, it may be sufficient to determine the response ofa single particle to a particular optical pattern. Thus, information maybe derived about the particle merely from the fact that the particlemoved, or moved in a particular way or by a particular amount. Thatinformation may be obtained irrespective of the presence or absence ofother particles. In yet other applications, it is desirable to separatetwo or more particles. In that case, by comparing the position of theparticles relative to each other such as in FIG. 12A versus 12B,information regarding the particle may be obtained. Having determinedwhich particle is the desired particle, assume for purposes ofdiscussion to be particle B, the particle may then be separated from theother particles. As shown in FIG. 12C, an optical tweezer intensityprofile 304 may be used to capture and remove particle B. Alternatively,as will be discussed in connection with FIGS. 14-19, the selectedparticle may be removed by other means, such as by fluidic means.

By utilizing a property of the particle, such as the optical dielectricconstant, the light forces serve to identify, select, characterizeand/or sort particles having differences in those attributes. Exposureof one or more particles to the optical force may provide informationregarding the status of that particle. No separation of that particlefrom any other particle or structure may be required. In yet otherapplications, the application of the optical force causes a separationof particles based upon characteristics, such that the separationbetween the particles may result in yet further separation. The modes offurther separation may be of any various forms, such as fluidicseparation, mechanical separation, such as through the use of mechanicaldevices or other capture structures, or optically, such as through theuse of an optical tweezer as shown in FIG. 12C, by application of amoving optical gradient, or by any other mode of removing or separatingthe particle, e.g., electromagnetic, fluidic or mechanical.

FIGS. 13A, 13B and 13C show potential energy as a function of distancefor one exemplary mode of operation. The figures show particle 1 andparticle 2 displaced in the x dimension relative to one another. Thephysical positioning of the two particles would typically be in the sameplane, e.g., the same vertical plane. The figures show the potentialenergy of the particle. In FIG. 13A, particle 1 310 is subject to lightintensity pattern creating the potential energy profile 314. Particle 2312 is subject to the same light intensity pattern but is subject to thesecond potential energy profile 316. The second potential energy profile316 is different from the first potential energy profile 314 because thedielectric constants are different between particle 1 310 and particle 2312. In FIG. 5A, the light intensity pattern is moving toward the right.As the potential energy profiles 314, 316 move to the right, theparticles 310, 312 experience different forces. Particle 1 310 willexperience a smaller force as compared to particle 2 312, as depicted bythe size of the arrows adjacent the particles. The force experienced bythe particles is proportional to the spatial derivative of the potentialenergy. Thus, particle 2 312 being on a relatively “steeper” portion ofthe potential energy “wave” would be subject to a larger force. In FIG.5A, the translation speed of the potential energy waves may be set to belarger than the speed at which particle 1 310 may move forward throughthe medium in which it is located. In that event, particle 1 310 may besubject to a force toward the left, FIG. 13A showing an arrow depictingthe possible backward or retrograde motion of particle 1 310. Thepotential energy wells have a minimum 318 into which the particles wouldsettle, absent motion or translation of the potential energy patterns314, 316.

FIG. 13B shows particle 1 310 and particle 2 312 subject to the firstpotential energy 314 and second potential energy 316, respectively. Asthe potential energy patterns 314, 316 translate to the right, theparticles 310, 312 are subject to a force to the right, though indifferent amounts as depicted by the relative size of the arrows. FIG.13C shows the potential energy profiles 314, 316 after the potentialenergy profiles of FIG. 13B have been moved so as to place the potentialenergy maximum between particle 1 310 and particle 2 312. By “jerking”the intensity profiles 314, 316 forward quickly, particle 1 310 is thenlocated on the “backside” of the potential energy “wave”, and would besubject to a force to the left. The path of motion is then shown by thedashed arrow from particle 1 310. In contrast, particle 2 312 remains onthe “front side” of the potential energy wave 316 and is subject to aforce to the right. The effect of this arrangement is to cause furtherphysical separation between particle 1 310 and particle 2 314. Thepotential energy profiles 314, 316 must be moved forward quickly enoughsuch that the potential energy maximum is located between the particlesto be separated, as well as to insure that the particle on the“backside” of the potential energy wave is caused to move away from theparticle on the “front side” of the wave.

The apparatus and methods of these inventions utilize optical forces,either alone or in combination with additional forces, to characterize,identify, select and/or sort material based upon different properties orattributes of the particles. The optical profiles may be static, thoughvary with position, or dynamic. When dynamic, both the gradient fieldsas well as the scattering forces may be made to move relative to theparticle, medium containing the particle, the support structurecontaining the particle and the medium. When using a moving opticalgradient field, the motion may be at a constant velocity (speed anddirection), or may vary in a linear or non-linear manner.

The optical forces may be used in conjunction with other forces.Generally, the optical forces do not interfere or conflict with theother forces. The additional forces may be magnetic forces, such asstatic magnetic forces as generated by a permanent magnet, or dynamicmagnetic forces. Additional electric forces may be static, such aselectrostatic forces, or may be dynamic, such as when subject toalternating electric fields. The various frequency ranges of alternatingelectromagnetic fields are generally termed as follows: DC isfrequencies much less than 1 Hz, audio frequencies are from 1 Hz to 50kHz, radio frequencies are from 50 kHz to 2 GHz, microwave frequenciesare from 1 GHz to 200 GHz, infrared (IR) is from 20 GHz to 400 THz,visible is from 400 THz to 800 THz, ultraviolet (UV) is from 800 THz to50 PHz, x-ray is from 5 PHz to 20 EHz and gamma rays are from 5 EHz andhigher (see, e.g., Physics Vade Mecum).) The frequency ranges overlap,and the boundaries are sometimes defined slightly differently, but theranges are always substantially the same. Dielectrophoretic forces aregenerated by alternating fields generally being in the single Hz to 10MHz range. For the sake of completeness, we note that dielectrophoreticforces are more electrostatic in nature, whereas optophoretic forces areelectromagnetic in nature (that is, comparing the frequency ranges isnot meant to imply that they differ only in their frequency.)Gravitational forces may be used in conjunction with optical forces. Byconfiguring the orientation of the apparatus, the forces of gravity maybe used to affect the actions of the particle. For example, a channelmay be disposed in a vertical direction so as to provide a downwardforce on a particle, such as where an optical force in the upwarddirection has been generated. The force of gravity takes intoconsideration the buoyancy of the particle. When a channel is disposedin the horizontal direction, other forces, e.g., frictional forces, maybe present. Fluidic forces (or Fluidics) may be advantageously utilizedwith optical forces. By utilizing an optical force to effect initialparticle separation, a fluidic force may be utilized as the mechanismfor further separating the particles. As yet another additional force,other optical forces may be applied against the particle. Any or all ofthe aforementioned additional forces may be used singly or incombination. Additionally, the forces may be utilized serially or may beapplied simultaneously.

FIGS. 14A and 14B show sorting of particles or objects from aone-dimensional source. As shown in FIG. 14A, particles 320 progress ina generally downward direction from a source in the direction of thearrow labeled particle flow. At junction 322, and possibly additionallybefore the junction 322, the particles are subject to an opticalseparation force. Those particles having a different response property,such as a different dielectric constant, may be separated from the lineof particles resulting in the separated particles 326. Those particleswhich are not separated continue on as the particles 324. FIG. 14B showsoptical cell sorting from a one-dimensional source. Cells 330 move in afluid flow in a direction from top to bottom as shown by the arrow. Thecells 330 are subject to an optical force in the region of junction 332.Selected cells 336 are deviated from the path of the original fluidflow. The remaining particles 334 continue on in the same direction asthe original fluid flow. It will be appreciated that the term “selected”or “non-selected” or similar terminology as used herein is meant to beillustrative, and not intended to be limiting.

The techniques of this invention may be utilized in a non-guided, i.e.,homogeneous, environment, or in a guided environment. A guidedenvironment may optionally include structures such as channels,including microchannels, reservoirs, switches, disposal regions or othervesicles. The surfaces of the systems may be uniform, or may beheterogeneous.

FIG. 15 shows a plan view of a guided structure including channels. Aninput channel 340 receives particles 342 contained within a medium. Anoptical force is applied in region 344. The optical force wouldpreferably be a moving optical gradient field. As the particles 342 movethrough the field 344, certain particles would be subject to a forcecausing them to move to the right in the channel as shown as particles346, yet other particles 348 would move to the left of the T channel. Byselection of the speed, orientation, periodicity, intensity and otherparameters of the optical force gradient, the particles may beeffectively separated.

The channels may be formed in a substrate or built upon some support orsubstrate. Generally, the depth of the channel would be on the order offrom substantially 1 to substantially 2 diameters of the particle. Formany biological cell sorting or characterization applications, the depthwould be on the order of 10 to 20 μm. The width of the channelsgenerally would be on the order of from substantially 2 to substantially8 diameters of the particle, to allow for at least one optical gradientmaximum with a width of the order of the particle diameter up to four ormore optical gradient maxima with a width of the order of the particlediameter. For many biological cell sorting or characterizationapplications, the width would be of the order of 20 to 160 micrometers.The channels may have varying shapes, such as a rectangular channelstructure with vertical walls, a V-shaped structure with intersectingnon-planar walls, a curved structure, such as a semicircular orelliptical shaped channel. The channels, or the substrate or base whenthe channel was formed within it, may be made of various materials. Forexample, polymers, such as silicon elastomers (e.g., PDMS), gels (e.g.,Agarose gels) and plastics (e.g., TMMA) may be utilized: glass, andsilica are other materials. For certain applications, it may bedesirable to have the support material be optically transparent. Thesurfaces may be charged or uncharged. The surface should have propertieswhich are compatible with the materials to be placed in contacttherewith. For example, surfaces having biological compatibility shouldbe used for biological arrays or other operations.

Various forms of motive force may be used to cause the particles,typically included within a fluid, to move within the system.Electroosmotic forces may be utilized. As known in the art, variouscoatings of the walls or channels may be utilized to enhance or suppressthe electroosmotic effect. Electrophoresis may be used to transportmaterials through the system. Pumping systems may be utilized such aswhere a pressure differential is impressed across the inlet and outletof the system. Capillary action may be utilized to cause materials tomove through the system. Gravity feeding may be utilized. Finally,mechanical systems such as rotors, micropumps, centrifugation may beutilized.

FIG. 16 shows an “H” channel structure for sorting of particles. TheH-shaped structure has two inlets and two outlets. The inlet 350receives both fluid and the subject particles 352 to be sorted. Fluid isinput in the second input arm of the H channel. The main or connectingchannel 356 receives the fluid flow from both inputs. In the connectingchannel 356, the particles 354 will flow through the connecting channeland be subject to the optical sorting force 358. At that stage, theparticles are then separated based upon the differentiating parameter,such as the particle's dielectric constant. The particles being movedfrom the primary stream move as particles 360 to one output. Theparticles 362 which are not diverted by action of the optical force 358continue to the left hand outlet 364. Laminar flow within the systemwill cause the particles 354 to move through the main channel 356, andif the channel width is large enough, will tend to cause the particles354 to flow relatively closer to the wall nearer the input. The sortingprocess then consists of diverting the particle from the laminar flowadjacent the left wall to the laminar flow which will divert to theright hand output.

FIG. 17 shows a wide channel structure for particle separation. Input370 receives the particles 372 in a fluidic medium. The particles aresubject to an optical sorting force 374, whereupon the divertedparticles 378 flow toward outlet 382 and particles 376 flow towardoutlet 380.

FIG. 18 shows an X-channel structure for sorting. Input 390 receivesparticles 392 in a fluidic medium. Second input 394 received fluid. Theparticles 392 are then subject to an optical sorting force 396. Divertedparticles 402 flow to exit 404. Particles 398 flow to exit 400.

FIG. 19 is a perspective drawing of a two-dimensional sorting system.The source inflow of cells 410 intersect with an optical sorting forcealong line 412. The sorting force 412 results in an outflow of targetcells 414 in one-dimension, typically in one plane, and an outflow ofnon-target cells 416 in another plane. The plane of outflow of targetscells 414 is non-coplanar with the plane of outflow of non-target cells416.

FIG. 20 shows an arrangement comprising a three-dimensional cell sortingarrangement. A volume 420, most preferably a substantiallythree-dimensional volume, though possibly a volume of lower effectivedimensionality, contains particles 422. An optical force gradient 428 isgenerated within the volume 420 to effect particle sorting. Oneembodiment for generating the optical field gradient 428 is to interferefirst beam 424 with a second beam 426. The first beam 424 and secondbeam 426 interfere and generate the force pattern 428. As shown, a firstparticle 430 is subject to a force in a direction from bottom to top,whereas a second particle 432 is subject to a force from top to bottom.Alternately, the optical pattern 428 may cause forces on particles 430,432 in the same direction, but with differing amounts of force.

FIG. 21 shows an embodiment having multiple degrees of freedom,preferably three degrees of freedom. The volume 440 contains particles442 which are disposed adjacent a surface, near the inwardly disposedsurface of mirror 450. An optical gradient force 444 is generated whichcauses selected ones of the particles 446 at the surface to be movedinto the volume 440 such as particle 446. The optical force gradient 444may be generated by shining an optical beam 448 onto a mirror 450, whichcauses interference between the beam 448 and its reflected beam.

FIG. 22 shows a multi-dimensional system in which a volume 450 isutilized to separate particles. First particles 452 are disposedadjacent the surface of the slide 454. A light intensity pattern 456causes displacement of selected particles. Those displaced particles maythen be attached to a sticky or adhesive mat 460 and comprises particles458.

FIG. 23 shows a plan view of a complex channel based system for sorting,characterization or classification. An input 470 leads through channel472 to a first optical sorting region 474. The sorting at a givenchannel is as described, before. The output of the sorting results in afirst set of particles 478 and a second set of particles 476. The firstset of particles 478 flows to the second optical sorting region 480. Asbefore, the particles are sorted into first particles 484 and secondparticles 482. A next optical sorting region 486 results in the outputof sorted particles, the first output 488 and second output 490 thenleading to further collection, counting or analysis. In one aspect, thecomplex system may include one or more recycle or feedback tabs 490. Asshown, the output from the optical force region 492 includes output 7but also a recycle path 494 leading to the input 496 coupling to thechannel 472. Such a recycle system might be used in an enrichmentsystem.

The systems described herein, and especially a more complex system, mayinclude various additional structures and functionalities. For example,sensors, such as cell sensors, may be located adjacent various channels,e.g., channel 742. Various types of sensors are known to those skilledin the art, including capacitive sensors, optical sensors and electricalsensors. Complex systems may further include various holding vessels orvesicles, being used for source materials or collection materials, or asan intermediate holding reservoir. Complex systems may further includeamplification systems. For example, a PCR amplification system may beutilized within the system. Other linear or exponential biologicalamplification methods known to those skilled in the art may beintegrated. Complex systems may further include assays or otherdetection schemes. Counters may be integrated within the system. Forexample, a counter may be disposed adjacent an output to tally thenumber of particles or cells flowing through the output. The systems ofthe instant invention are useable with microelectromechanical (MEMs)technology. MEMs systems provide for microsized electrical andmechanical devices, such as for actuation of switches, pumps or otherelectrical or mechanical devices. The system may optionally includevarious containment structures, such as flow cells or cover slips overmicrochannels.

A computerized workstation may include a miniaturized sample stationwith active fluidics, an optical platform containing a laser (e.g., anear infrared laser for biological applications) and necessary systemhardware for data analysis and interpretation. The system may includereal-time analysis and testing under full computer control.

The inventions herein may be used alone, or with other methods of cellseparation. Current methods for cell separation and analysis includeflow cytometry, density gradients, antibody panning, magnetic activatedcell sorting (“MACS™”), microscopy, dielectrophoresis and variousphysiological and biochemical assays. MACS separations work only withsmall cell populations and do not achieve the purity of flow cytometry.Flow cytometry, otherwise known as Fluorescent Activated Cell Sorting(“FACS™”) requires labeling.

In yet another aspect, the systems of the present invention mayoptionally include sample preparation steps and structure for performingthem. For example, sample preparation may include a preliminary step ofobtaining uniform size, e.g., radius, particles for subsequent opticalsorting.

The systems may optionally include disposable components. For example,the channel structures described may be formed in separable, disposableplates. The disposable component would be adapted for use in a largersystem that would typically include control electronics, opticalcomponents and the control system. The fluidic system may be included inpart in the disposable component, as well as in the non-disposablesystem components.

FIG. 24 shows a system for optical sorting based upon a physicalparameter of the object, such as deformability. An optical gradient 500may illuminate particles 502, 504. Particle 504 is more deformable thanparticle 502. As a result, given the periodicity of the optical forcepattern 500, the deformable particle 504 may be subject to a relativelylarger force, and move more under the optical field 500. Preferably, theoptical field 500 is a moving optical gradient field. Alternatively, theparticles 502, 504 may be subject to the optical force 500, and thestructure of the particles 502, 504 monitored. In that way, by observingthe deformability of the particles, relative to the light pattern 500,the particles may be identified, classified or otherwise sorted.

FIG. 25 shows a method for sorting particles based upon size. An opticalintensity pattern 510 illuminates larger particle 512 and smallerparticle 514. The differently sized particles 512, 514 are subject todifferent forces. Where, for example, larger particle 512 spans two ormore intensity peaks of the optical gradient 510, the particle may haveno net force applied to it. In contrast, the smaller particle 514 whichhas a size smaller than the period of the optical intensity pattern 510may be subject to a relatively larger force. By selection of the periodof the optical pattern 510 relative to the size of particles to besorted, the system may effectively sort based upon size. In one method,a set of particles may be subject to an increasing period of the lightintensity, such that smaller particles are removed first, followed bythe relatively larger particles at a later time. In this way, particlesmay be effectively sorted by size.

Methods for Reducing or Modifying Forces

The system and methods may include various techniques for reducing orotherwise modifying forces. Certain forces may be desirable in certainapplications, but undesirable in other applications. By selecting thetechnique to reduce or minimize the undesired forces, the desired forcesmay more efficiently, sensitively and specifically sort or identify thedesired particles or conditions. Brownian motion of particles may be anundesired condition for certain applications. Cooling of the system mayresult in a reduced amount of Brownian motion. The system itself may becooled, or the fluidic medium may be cooled.

Yet another force which may be undesired in certain applications isfriction or other form of sticking force. If surface effects are to beminimized, various techniques may be utilized. For example, acounterpropagating beam arrangement may be utilized to capture particlesand to remove them from contact with undesired surfaces. Alternatively,the particles may be levitated, such as through the use of reflectedlight (see, e.g., FIG. 4, mirror 108). FIG. 4A shows an alternativearrangement for particle levitation. Opposing forces of twocounter-propagating optical beams can be used to levitate a particle toreduce surface friction drag.

Yet other techniques exist for addressing friction, stiction,electrostatic and other surface interactions which may interfere withthe mobility of cells and/or particles. For example, surfaces may betreated, such as through the use of covalent or non-covalentchemistries, which may moderate the frictional and/or adhesion forces.Surfaces may be pretreated to provide better starting surfaces. Suchpretreatments may include plasma etching and cleaning, solvent washesand pH washes, either singly or in combination. Surfaces may also befunctionalized with agents which inhibit or minimize frictional andadhesive forces. Single or multi-step, multi-layer chemistries may beutilized. By way of example, a fluorosilane may be used in a singlelayer arrangement which renders the surface hydrophobic. A two-step,two-layer chemistry may be, for example, aminopropylsilane followed bycarboxy-PEG. Teflon formal coating reagents such as CYTOP™ or Parylene™can also be used. Certain coatings may have the additional benefit ofreducing surface irregularities. Functional groups may, in certaincases, be introduced into the substrate itself. For example, a polymericsubstrate may include functional monomers. Further, surfaces may bederivitized to provide a surface which is responsive to other triggers.For example, a derivatized surface may be responsive to external forces,such as an electric field. Alternatively, surfaces may be derivatizedsuch that they selectively bind via affinity or other interactions.

Yet another technique for reducing surface interactions is to utilize abiphasic medium where the cells or particles are kept at the interface.Such aqueous polymer solutions, such as PEG-dextran partition into twophases. If the cells partitioned preferentially into one of the layers,then under an optical gradient the cells would be effectively floatingat the interface.

Methods for Enhancing or Changing the Dielectric Constant

Optionally, the particles to be subject to the apparatus and methods ofthese inventions may be either labeled or unlabeled. If labeled, thelabel would typically be one which changes or contributes to thedielectric constant of the particle or new particle (i.e., the initialparticle and the label will act as one new particle). For example, agold label or a diamond label would effectively change most typicaldielectric constants of particles.

Yet other systems may include an expressible change in dielectricconstant. For example, a genetic sequence may exist, or be modified tocontain, an expressible protein or other material which when expressedchanges the dielectric constant of the cell or system. Another way totune the dielectric constant of the medium is to have a single medium ina fluidic chamber where the dielectric constant can be changed bychanging the temperature, applying an electric field, applying anoptical field , etc. Other examples would be to dope the medium with ahighly birefringent molecule such as a water-soluble liquid crystal,nanoparticles, quantum dots, etc. In the case of birefringent molecules,the index of refraction that the optical beam will see can be altered bychanging the amplitude and direction of an electric field.

Methods for Increasing Sensitivity

Maximizing the force on a particle for a given intensity gradientsuggests that the difference in dielectric constant between the particleand medium should be maximized. However, when sensitivity is required inan application, the medium should be selected such that the dielectricconstant of the medium is close to the dielectric constant of theparticle or particles to be sorted. By way of example, if the particlepopulation to be sorted has dielectric constants ranging from 1.25 to1.3, it would be desirable to choose a dielectric constant which isclose to (or even within) that range. For cells, a typical range ofdielectric constants would be from 1.8 to 2.1. By close, a dielectricconstant within 10% or, more particularly, within 5%, would beadvantageous. While the absolute value of the magnitude of the force onthe particle population may be less than in the case where thedielectric constant differs markedly from the dielectric constant of themedium, the difference in resulting motion of the particles may belarger when the dielectric constant of the medium is close to the rangeof dielectric constants of the particles in the population. Whileutilizing the increased sensitivity of this technique at the outset,once the separation begins, the force may be increased by changing thedielectric constant of the medium to a more substantial difference fromthe dielectric constants of the particle or particle collection. Asindicated, it is possible to choose the dielectric constant of themedium to be within the range of dielectric constants of the particlepopulation. In that instance, particles having a dielectric constantabove the dielectric constant of the medium will feel a force in onedirection, whereas those particles having a dielectric constant lessthan the dielectric constant of the medium will feel a force moving inthe opposite direction.

Scattering Force Systems

It is possible to utilize the scattering force, either alone or incombination with the optical gradient force, such as supplied by amoving optical field gradient, for separation of particles. FIG. 26shows the before and after depiction of a system including a laser 520and a lens 522 which collimates the optical beam. A capillary 524receives the illumination, preferably along its axis. A set ofparticles, first particles 526 and second particles 528, are illuminatedby the light beam and are subject to different scattering forcesdepending upon their different scattering properties. Because of thedifferent forces, first particles 526′ move a shorter distance thansecond particles 528′, as shown in the second drawing. In this way,optical forces, particularly optical scattering forces, may be utilizedto separate particles.

FIGS. 27A, 27B and 27C depict a scattering force switch. A first input530 couples via a channel to a first output 536. The second input 532couples to a second output 538 via a channel. The two channels overlapby providing a fluidic connection between them. In operation, a particleentering in input 1 530 may be switched by a scattering force switch 540by deviating the particle from the channel coupled to input 1 530 to thechannel containing output 2 538. Scattering force switches may be usedin conjunction with the optical gradient force systems, especially themoving optical gradient force systems described herein.

Static Systems

FIG. 28 shows a system for the measurement of dielectric constants ofparticles. A particle 558 having a dielectric constant may be subject todifferent media having different dielectric constants. As shown, a firstvessel 550, a second vessel 552, and so on through an end vessel 554contain a medium having different dielectric constants ∈₁ ∈₂, . . .∈_(n), respectively. By illuminating the particle 558 with an opticalgradient force 556, and observing the motion, the dielectric constant ofthe particle may be determined. If the dielectric constant of the mediumis equal to the dielectric constant of the particle then no force isimposed by the optical illumination 556. In contrast, if there is adifference between the dielectric constant of the particle and thedielectric constant of the medium, an optical force will be imposed onthe particle by the optical illumination 556. Different dielectricconstant media may be supplied as shown in FIG. 28, namely, where aplurality a vessels 550, 552 . . . 554 are provided. Alternately, aparticle may be subject to a varying dielectric constant over time, suchas through use of a titration system. In on implementation, thetitration may be accomplished in a tube containing the particle byvarying the dielectric constant of the fluid over time, such as bymixing fluids having different dielectric constants, preferably at theinlet to the tube, or by providing a varying dielectric constantprofile, such as a step profile. Additionally, the dielectric constantof a particle may be approximated by interpolation, such as where two ormore data points are obtained regarding the force on the particle indifferent media, and then the expected dielectric constant in which noforce is present may be determined.

FIG. 29 shows a static system in which separation may occur. A lightpattern 560 illuminates first particle 562 and second particle 564. Ifthe dielectric constant of the first particle 562 is less than thedielectric constant of the medium, then the particle moves toward anarea of lower intensity. In contrast, if the second particle 564 has adielectric constant which is greater than the dielectric constant of themedium, the particle will move toward the region of higher intensity. Asa result, the first particle 562 and second particle 564 are subject toforces in opposite directions. Given the proximity shown, they wouldmove away from one another.

FIG. 30 shows a system for the use of a plurality of optical tweezers,preferably in an array, such as to move materials. A substrate 570 maycontain one or more sites 572 on which materials may be placed. Thematerials may comprise particles, cells, or any other material to beselected or moved. An optical tweezer array may selectively movematerials, such as those shown as light circles 576, and move thosematerials to yet another portion of the substrate 570, such as array574. Alternatively, the optical tweezer array may illuminate the entirearray 572, and then selectively move the materials as to which theoptical tweezer array provides sufficient force to cause separation ofthe particles 576, 578 from the array 572 on the substrate 570. Forexample, the particles may have attachment mechanisms, such ascomplimentary nucleic acids, which selectively bind them to thesubstrate 570.

FIG. 31 shows a graph of molar extinction coefficient as a function ofwavelength for hemoglobin-O₂ absorption. For certain sortingapplications, it may be desirable to select a wavelength forillumination which is at or near a peak of absorption. For example, itmay be desirable to choose a wavelength at the 500,000 molar extinctioncoefficient peak. Alternatively, it may be desirable to choose asecondary peak, e.g., the peak at substantially 560 nm or atsubstantially 585 nm.

The first setup is a moving fringe workstation for optophoresisexperiments. A high power, 2.5 watt, Nd-YAG laser (A) is the near IR,1064 nm wavelength, light source. The fringe pattern is produced bydirecting the collimated laser beam from the mirror (1) through theMichelson interferometer formed by the prism beam splitter (2) and thecarefully aligned mirrors (3). A variable phase retarder (4) causes thefringe pattern to continuously move. This fringe pattern is directed bythe periscope (5) through the telescope (5 a) and (5 b) to size thepattern to fill the back focal plane of the microscope objective, andthen is directed by the dichroic beam splitter (6) through a 20×microscope objective (7) to produce an image of the moving fringepattern in the fluidic chamber holding the sample to be sorted. Asecond, 60× microscope objective (8) images the flow cell onto a CCDcamera to provide visualization of the sorting experiments. Afiber-optic illuminator (9) provides illumination, through the dichroicbeam splitter (6), for the sample in the fluidic chamber. The fluidicchamber is positioned between the two microscope objectives by means ofan XYZ-translation stage.

It will be appreciated by those skilled in the art that there are anynumber of additional or different components which may be included. Forexample, additional mirrors or other optical routing components may beused to ‘steer’ the beam where required. Various optical components forexpanding or collimating the beam may be used, as needed. In the set-upimplementing FIG. 5, the laser used additional mirrors to steer thelaser beam into the spatial filter, which that produced a wellcollimated Gaussian beam that is then guided to the Michelsoninterferometer.

The second setup is a workstation for measuring and comparing thedielectric properties of cells and particles at near IR opticalfrequencies, using a 600 mW, ultra-low noise Nd-YAG laser (B) as a lightsource. The remainder of the optical setup is similar to the movingfringe workstation, except there is no interferometer to produce movingfringes. Instead a single, partially focused illumination spot is imagedwithin the fluidic chamber. The interaction of cells with thisillumination field provides a measurement of the dielectric constant ofthe cells at near IR optical frequencies.

Exemplary Applications

High Throughput Biology

The methods and apparatus herein permit a robust cell analysis systemsuitable for use in high throughput biology in pharmaceutical and lifesciences research. This system may be manufactured using higherperformance, lower cost optical devices in the system. A fullyintegrated high throughput biology, cell analysis workstation issuitable for use in drug discovery, drug discovery, toxicology and lifescience research. These systems may utilize advanced opticaltechnologies to revolutionize the drug discovery process and cellularcharacterization, separation and analysis by integrating optophoresistechnology into devices for the rapid identification, selection andsorting of specific cells based on their innate properties, includingtheir innate optical dielectric properties. In addition, since thetechnology is based on the recognition of such innate properties, labelsare not required, greatly simplifying and accelerating the testingprocess. The lasers employed are preferably in thebiologically-compatible infrared wavelengths, allowing precise cellcharacterization and manipulation with little or no effect on the cellitself. The technology is suited to the post-genomics era, where theinteraction of the cell's molecular design/make-up (DNA, RNA andproteins) and the specific cellular changes (growth, differentiation,tissue formation and death) are of critical importance to the basicunderstanding of health and disease.

The Optophoresis technology changes the nature of cell-based assays.Applications would include all methods of cellular characterization andsorting. The technology also offers diverse applications in the areas ofmolecular and cellular physiology. Optophoresis technology addressesfundamental properties of the cell itself, including its opticaldielectric properties. The optophoretic properties of the cell changefrom cell type to cell type, and in response to external stimuli. Theseproperties are reflective of the overall physiologic status of the cell.Active cells have dielectric properties that are different from restingcells of the same type. Cancer cells have different optophoreticproperties than their normal counterparts. These cellular properties canalso be used effectively in drug discovery and pharmaceutical research,since nearly all drugs are targeted ultimately to have direct effects oncells themselves. In other words, drugs designed to effect specificmolecular targets will ultimately manifest their effects on cellularproperties as they change the net dielectric charge of the cell.Therefore, rapid screening of cells for drug activity or toxicity is anapplication of the technology, and may be referred to as High ThroughputBiology. Other main applications include drug discovery andpharmaceutical research.

The Human Genome Project and other associated genome programs willprovide enormous demand for improved drug development and screeningtechnologies. Sophisticated cellular approaches will be needed forcost-effective and functional screening of new drug targets. Likewise,information from the genome projects will create demand for improvedmethods of tissue and organ engineering, each requiring access to wellcharacterized cellular materials. Moreover, optical technology from theinformation and telecommunications industry will provide the systemhardware for improved optical cell selection and sorting. Theprice/performance ratios for high powered near infrared and infraredlasers originally developed for telecommunications applications continueto improve significantly. In addition, solid-state diode lasers may beused having a variety of new wavelengths, with typically much higherpower output than older versions. Vertical Cavity Surface EmittingLasers (“VCSELs”) provide arrays of diode lasers at very reasonablecosts with increasing power output.

A computerized Workstation may be composed of a miniaturized samplestation with active fluidics, an optical platform containing a nearinfrared laser and necessary system hardware for data analysis andinterpretation. The system includes real-time analysis and testing underfull computer control. Principal applications of the technology includecell characterization and selection, particularly for identifying andselecting distinct cells from complex backgrounds.

Importantly, unlabelled, physiologically normal, intact test cells willbe employed in the system. The sample is quickly analyzed, with thecells classified and sorted by the optical field, thereby allowingcharacterization of drug response and identify toxicity or othermeasures of drug efficacy. Characterizing the cellular optophoreticproperties uniquely associated with various drug testing outcomes anddisease states is a part of this invention. Identification of thesenovel parameters constitutes useful information.

An integrated system may, in various aspects, permit: theidentification, selection and separation of cells without the use oflabels and without damaging the cells; perform complex cell analysis andseparation tasks with ease and efficiency; observe cells in real time asthey are being tested and manipulated; establish custom cell sortingprotocols for later use; isolate rare cells from complex backgrounds;purify and enrich rare cells (e.g. stem cells, fragile cells, tumorcells); more easily link cell phenotype to genotype; study cell—cellinteractions under precise and optical control; and control sampleprocessing and analysis from start to finish.

The technology offers a unique and valuable approach to buildingcellular arrays that could miniaturize current assays, increasethroughput and decrease unit costs. Single cell (or small groups ofcells) based assays will allow miniaturization, and could allow moredetailed study of cell function and their response to drugs and otherstimuli. This would permit cellular arrays or cell chips to performparallel high-throughput processing of single cell assays. It could alsopermit the standardization of cell chip fabrication, yielding a moreefficient method for creation of cell chips applicable to a variety ofdifferent cells types.

Mammalian cell culture is one of the key areas in both research (e.g.,discovery of new cell-produced compounds and creation of new cell linescapable of producing specific proteins) and development (e.g.,developing monoclonal cell lines capable of producing highly specificproteins for further research and testing). Mammalian cell culture isalso a key technology for the production of new biopharmaceuticals on acommercial scale.

Once researchers have identified drug targets, compounds or vaccines,mammalian cell culture is an important technology for the production ofquantities necessary for further research and development. There arecurrently more than 70 approved biotechnology medicines and more than350 such compounds in testing, targeting more than 200 diseases.

Optical cell characterization, sorting and analysis technologies couldbe useful in selecting and separating lines of mammalian cells accordingto whether they produce a new protein or biopharmaceutical compound andaccording to the yield of the protein or compound. Cell yield is a keyfactor in determining the size of the plant a manufacturer must build toproduce commercial quantities of a new biotechnology drug.

We turn now to more specific discussions of applications. First, weaddress separation applications, and second, address monitoringapplications.

Separation Applications

White cells from red cells. White blood cells are the constituents ofblood which are responsible for the immune response as compared with redcells which transport oxygen through the body. White cells need to beremoved from red cells prior to transfusion for better tolerance and todecrease infection risks. It is also often important to remove red cellsin order to obtain enriched populations of white cells for analysis ormanipulation. Optophoresis can allow the separation of these twodistinct cell populations from one another for use in applications wherea single population is required.

Reticulocytes from mature red blood cells. Reticulocytes, which areimmature red blood cells normally found at very low levels can beindicators of disease states when they are found at increased levels.This application would use optophoresis for the separation andenumeration of the levels of reticulocytes from whole blood.

Clinical Care Applications, e.g., Fetal stem cells from maternalcirculation. The Clinical Care applications include cell-basedtreatments and clinical diagnostics. The successful isolation of fetalcells from maternal blood represents a source of fetal DNA obtainable ina non-invasive manner. A number of investigators worldwide have nowdemonstrated that fetal cells are present in the maternal circulationand can be retrieved for genetic analysis. The major current challengesin fetal cell isolation include selection of the target fetal cell type,selection and isolation of the cells and the means of genetic analysisonce the cells are isolated. Using a maternal blood sample, the systemcan identify the rare fetal cells circulating within the mother's bloodand to permit the diagnosis of genetic disorders that account for up to95% of prenatal genetic abnormalities, e.g., Down's Syndrome. Cell-basedtreatments refer to procedures similar to diagnostic procedures, but forwhich the clinical purpose is somewhat broader. During pregnancy, asmall number of fetal cells enter the maternal circulation. By purifyingthese cells using optophoresis prenatal diagnosis of a variety ofgenetic abnormalities would be possible from a single maternal bloodsample.

Clinical Care Applications, e.g., Stem Cell Isolation. The purpose ofstem cell isolation is to purify stem cells from stem cell grafts fortransplantation, i.e., to remove T-cells in allogeneic grafts (where thedonor and the recipient are not the same person) and cancer cells inautologous grafts (where the donor and the recipient are the sameperson). Currently stem cell technologies suffer from several drawbacks.For example, the recovery efficiency of stem cells obtained usingcurrently available systems is on the order of 65-70%. In addition,current methods do not offer the 100% purity which is beneficial intransplant procedures.

Tumor cells from blood. Minimal Residual Disease (MRD) Testing TheNational Cancer Institute (NCI) estimates that approximately 8.4 millionAmericans alive today have a history of cancer, and that over 1.2million new cancer cases were diagnosed in 2000. The NCI also estimatesthat since 1990 approximately 13 million new cancer cases werediagnosed, excluding noninvasive and squamous cell skin cancers.Optophoresis technology addresses some of the key unmet needs for bettercancer screening, including: accurate, reproducible and standardizedtechniques that can detect, quantify and characterize disseminatedcancer cells; highly specific and sensitive immunocytologicaltechniques; faster speed of cell sorting; and techniques that cancharacterize and isolate viable cancer cells for further analysis.

Cancer cells may be found in low numbers circulating in the blood ofpatients with various forms of that disease, particularly whenmetastasis has occurred. The presence of tumor cells in the blood can beused for a diagnosis of cancer, or to follow the success or failure ofvarious treatment protocols. Such tumor cells are extremely rare, so ameans of enrichment from blood such as optophoresis would need to beemployed in order to have enough cells to detect for accurate diagnosis.Another application for optophoresis in this regard would be to removetumor cells from blood or stem cell products prior to them being used toperform an autologous transplant for a cancer patient.

Fetal stem cells from cord blood. The umbilical cord from a newborngenerally contains blood which is rich in stem cells. The cord bloodmaterial is usually discarded at birth; however, there are both academicand private concerns who are banking cord blood so that such discardedmaterial can be used for either autologous or allogenic stem cellreplacement. Enrichment of the cord blood stem cells by optophoresiswould allow for a smaller amount of material to be stored, which couldbe more easily given back to the patient or another host.

Adult stem cells from liver, neural tissue, bone marrow, and the Like.It is becoming increasingly clear that many mature tissues have smallsubpopulations of immortal stem cells which may be manipulated ex vivoand then can be reintroduced into a patient in order to repopulate adamaged tissue. Optophoresis can be used to purify these extremely rareadult stem cells so that they may be used for cell therapy applications.

Islet cells from pancreas. It has been proposed that for persons withdiabetes resulting from lack of insulin production, the insulinproducing beta islet cells from a healthy pancreas could be transplantedto restore that function to the diabetic person. These cells make uponly a small fraction of the total donor pancreas. Optophoresis providesa method to enrich the islet cells and would be useful for preparationof this specific type of cell for transplantation.

Activated B or T cells. During an immune response either T or B whitecell subsets which target a specific antigen become active. Thesespecific activated cells may be required as separate components for usein ex vivo expansion to then be applied as immunotherapy products or tobe gotten rid of, since activated B or T cells can cause unwanted immunereactions in a patient such as organ rejection, or autoimmune diseasessuch as lupus or rheumatoid arthritis. Optophoresis provides a method toobtain activated cells either to enrich and give back to a patient or todiscard cells which are causing pathological destruction.

Dendritic cells. Dendritic cells are a subset of white blood cells whichare critical to establishing a T-cell mediated immune response. Biotechand pharmaceutical companies are working on ways to harvest dendriticcells and use them ex vivo in conjunction with the appropriate antigento produce a specific activated T cell response. Optophoresis wouldallow isolation of large numbers of dendritic cells for such work.

HPRT-cells. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is anenzyme which exits in many cells of the blood and is involved in thenucleoside scavenging pathway. Persons who have high mutation rates dueto either endogenous genetic mutations or exogenous exposure to mutagenscan be screened for HPRT lacking cells (HPRT-) which indicate a mutationhas occurred in this gene. Optophoresis following screening by compoundswhich go through the HPRT system can be used to easily select HPRT minuscells and quantitate their numbers.

Viable or mobile sperm cells. Approximately 12% of couples are unable toinitiate a pregnancy without some form of assistance or therapy. Inabout 30% these cases, the male appears to be singularly responsible. Inan additional 20% of cases, both male and female factors can beidentified. Thus, a male factor is partly responsible for difficultiesin conception in roughly 50% of cases. The number of women aged 15-44with impaired ability to have children is well over 6 million. Semenanalysis is currently performed using a variety of tests and is based ona number of parameters including count, volume, pH, viscosity, motilityand morphology. At present, semen analysis is a subjective and manualprocess. The results of semen analysis do not always clearly indicate ifthe male is contributing to the couple's infertility. Gradientcentrifugation to isolate motile sperm is an inefficient process (10 to20% recovery rate). Sperm selection is accomplished using eithergradient centrifugation to isolate motile sperm used in In UteroInsemination (IUI) and In Vitro Fertilization (IVF) or visual inspectionand selection to isolate morphologically correct sperm used in IVF andIntracytoplasmic Sperm Injection (ICSI). Each year in the U.S., 600,000males seek medical assistance for infertility.

One of the reasons for male infertility is the lack of high enoughpercentages of viable and/or mobile sperm cells. Viable and/or mobilesperm cells can be selected using optophoresis and by enriching theirnumbers, higher rates of fertilization can be achieved. This applicationcould also be used to select X from Y bearing sperm and vice versa,which would then be used selectively to induce pregnancies in animalapplications where one sex of animal is vastly preferred for economicreasons (dairy cows need to be female, while it is preferable for meatproducing cattle to be male for example).

Liposomes loaded with various compounds. A recent mode of therapeuticdelivery of pharmaceutical products is to use liposomes as the deliveryvehicle. It should be possible using optophoresis to separate liposomeswith different levels of drug in them and to enrich for those liposomesin which the drugs are most concentrated.

Tissue Engineering, e.g., Cartilage precursors from fat cells. Tissueengineering involves the use of living cells to develop biologicalsubstitutes for tissue replacements which can be used in place oftraditional synthetic implants. Loss of human tissue or organ functionis a devastating problem for a patient and family. The goal of tissueengineering is to design and grow new tissue outside the body that couldthen be transplanted into the body.

A recent report has demonstrated that cells found in human adiposetissue can be used ex vivo to generate cartilage which can be used as atransplant material to repair damage in human joints. Optophoresis canbe used to purify the cartilage forming cells from the other cells inadipose tissue for ex vivo expansion and eventual tissue engineeringtherapy.

Nanomanipulation of small numbers of cells. Recent miniaturization ofmany lab processes have resulted in many lab analyses being put ontosmaller and smaller platforms, evolving towards a “lab-on-a-chip”approach. While manipulation of biomolecules in solution has becomeroutine in such environments, manipulation of small numbers of cells inmicrochannel and other nano-devices has not been widely achieved.Optophoresis will allow cells to be moved in microchannels and directedinto the region with the appropriate processes on the chip.

Cellular organelles; mitochondria, nucleus, ER, microsomes. The internalconstituents of a cell consists of the cytoplasm and many organellessuch as the mitochondria, nucleus, etc. Changes in the numbers orphysical features of these organelles can be used to monitor changes inthe physiology of the cell itself. Optophoresis can allow cells to beselected and enriched which have particular types, morphologies ornumbers of a particular organelle.

Cow reticulocytes for BSE assays. It has been reported that a cellularcomponent of the reticulocyte, EDRF, is found at elevated levels in thereticulocytes of cows infected with BSE (bovine spongiformencephalopathy). Reticulocytes are generally found at low levels in theblood and therefore the use of optophoresis would allow their enrichmentand would increase the accuracy of diagnostic tests based on thequantitation of the EDRF mRNA or protein.

Monitoring

Growing/dividing cells vs. resting cells. Cells may be stimulated togrow by various growth factors or growth conditions. Most assays whichexist for cell growth require the addition of external labeling reagentsand/or significant time in culture before cell growth can bedemonstrated. By using optophoresis, cells which have begun to dividewill be identified, providing a rapid method for calculating how much ofa given cell population is in the growth phase. Cells in different partsof the cell cycle should have different optical properties and these maybe used to either sort cells based on where in the cycle they are aswell as to determine what fraction of the total cell population is ineach stage of the cell cycle.

Apoptotic cells. Cells which are undergoing programmed cell death orapoptosis can be used to identify specific drugs or other phenomenonwhich lead to this event. Optophoresis can be used to identify whichcells are undergoing apoptosis and this knowledge can be used to screennovel molecules or cell conditions or interactions which promoteapoptosis.

Cells with membrane channels open; change in membrane potentials. Theouter membrane of many types of cells contain channels which facilitatethe passage of ions and small molecules into and out of the cell.Movement of such molecules can lead to further changes in the cell suchas changes in electrical potential, changes in levels of secondmessengers, etc. Knowledge of these changes can be useful in drugscreening for compounds which modulate membrane channel activity.Optophoresis can be used to indicate when membrane channels are beingperturbed by exogenous compounds.

Live vs. dead cells. Many applications exist which require theidentification and quantitation of live versus dead cells. By usingoptophoresis dead cells can be identified and counted.

Virally infected cells. There are many diagnostic applications where itis important to measure cells which contain virus, including ones forCMV, HIV, etc. Optophoresis can be used to differentiate cells whichcontain virus from cells which do not.

Cells with abnormal nucleus or elevated DNA content. One of thehallmarks of a tumor cell is that it will contain either excess DNA,resulting in an abnormal size and/or shape to it's nucleus. By usingoptophoresis tuned to the nuclear content of a cell populations withabnormal amounts of DNA and/or nuclear structure may be identified andthis information can be used as a diagnostic or prognostic indicator forcancer patients.

Cells decorated with antibodies. A large selection of commerciallyavailable antibodies exists which have specificities to cellular markerswhich define unique proteins and/or types of cells. Many diagnosticapplications rely on the characterization of cell types by identifyingwhat antibodies bind to their surface. Optophoresis can be used todetect when a cell has a specific antibody bound to it.

Cells with bound ligands, peptides, growth factors. Many compounds andproteins bind to receptors on the surface of specific cell types. Suchligands may then cause changes inside the cell. Many drug screens lookfor such interactions. Optophoresis provides a means to monitor bindingof exogenous large and small molecules to the outside of the cell, aswell as measurement of physiological changes inside the cell as a resultof compound binding.

Bacteria for viability after antibiotic exposure. Microorganisms areoften tested for sensitivity to a spectrum of antibiotics in order todetermine the appropriate therapy to pursue to kill an infectiousorganism. Optophoresis can be used to monitor bacterial cells forviability and for cessation of growth following antibiotic exposure.

Drug screening on the NCI 60 panel. A panel of 60 tumor cell lines hasbeen established by the National Cancer Institute as a screening tool todetermine compounds which may have properties favorable to use aschemotherapeutic agents. It should be possible to use optophoresis toarray all 60 lines and then to challenge them with known and novelchemicals and to monitor the cell lines for response to the chemicals.

Cells for cytoskeletal changes. The cytoskeleton is a complex ofstructural proteins which keeps the internal structure of the cellintact. Many drugs such as taxol, vincristine, etc. as well as otherexternal stimuli such as temperature are known to cause the cytoskeletonto be disrupted and breakdown . Optophoresis provides a means to monitorpopulations of cells for perturbations in the cytoskeleton.

Beads with compounds bound to them, to measure interactions with thecell surface or with other beads. The interactions of microspheres withcells or other compounds has been used in a number of in vitrodiagnostic applications. Compounds may be attached to beads and theinteractions of the beads with cells or with beads with other compoundson them can be monitored by optophoresis.

Progenitor cell/colony forming assays. Progenitors are cells of a giventissue which can give rise to large numbers of more mature cells of thatsame tissue. A typical assay for measuring progenitor cells is to allowthese cells to remain in culture and to count how many colonies of theappropriate mature cell type they form in a given time. This type ofassay is slow and cumbersome sometimes taking weeks to perform. By usingoptophoresis to monitor the growth of a single cell, progenitorproliferation can be measured on a nano-scale and results should beobtained within a much shorter length of time.

Dose limiting toxicity screening. Almost all compounds are toxic at somelevel, and the specific levels of toxicity of compounds are identifiedby measuring at what concentration they kill living cells and organisms.By monitoring living cells with optophoresis as the dose of a compoundis slowly increased, the level at which optical properties indicative ofcell damage and/or death can be ascertained.

Monitor lipid composition/membrane fluidity in cells. The membranes ofall cells are composed of lipids which must maintain both the properdegree of membrane fluidity at the same time that they maintain basiccell membrane integrity. Optophoresis should be able to measure thefluidity of the membrane and to provide information on compounds andconditions which can change membrane fluidity, causing membranes to beeither more or less fluid.

Measure clotting/platelet aggregation. Components found in the bloodsuch as platelets and clotting proteins are needed to facilitate bloodclot formation under the appropriate circumstances. Clotting is oftenmonitored in order to measure disease states or to assess basic bloodphysiology. Optophoresis can provide information on platelet aggregationand clot formation.

Certain of the data reported herein were generated with the followingsetup. Optical gradient fields were generated using a Michelsoninterferometer and either a 150 mW, 812 nm laser (812 system) or a 2.5W, 1064 nm laser (1064 system). The 812 system used a 100× (1.25 NA) oilimmersion lens to focus the fringe pattern and to visualize the sample.The 1064 system used a 20× objective to focus the fringes and a 60×objective to visualize the sample. In general the sample cell was acoated microscope slide and/or coverslip that was sealed with Vaseline.Coverslip spacers controlled the height of the cell at approximately 150micrometers

Coating Of Surfaces; Rain-X™, Agarose, CYTOP, Fluorosilane Scatteringforces tend to push the particles or cells against the surface of thesample cell. Therefore, a number of surface coatings were evaluated tominimize nonspecific adhesion and frictional forces.Hydrophobic/hydrophilic and covalent/noncovalent surface treatments wereevaluated.

Covalent/Hydrophobic Glass slides and coverslips were treated withperfluoro-octyltrichlorosilane (Aldrich, Milwaukee, Wis.) using solutionor vapor deposition. Solution deposition was as follows: a 2-5% silanesolution in ethanol, incubate 30 minutes at room temperature, rinse 3times in ethanol and air dry. Vapor deposition involved applying equalvolumes of silane and water in separate microcentrifuge tubes andsealing in a vacuum chamber with the substrate to be treated. Heat to50° C., 15 hrs.

Noncovalent/Hydrophobic—A commercial water repellent containingpolysiloxanes, Rain-X, was applied according to the manufacturer'sinstructions.

A liquid Teflon, CYTOP (CTL-107M, Wilmington, Del.) was spun coatedusing a microfuge. The CYTOP was diluted to 10% in fluorooctane (v/v)and 50 microliters was pipetted and spun for 5 seconds. This wasrepeated a second time and then air dried.

Noncovalent/Hydrophilic—Agarose hydrogel coatings were prepared asfollows: melt 2% agarose in water, pipette 100 microliters to thesubstrate, spin for 5 seconds, bake at 37° C. for 30 minutes.

All of the coatings were effective when working with particles. TheCYTOP was more effective at preventing adhesion when working withbiological cells.

Separation By Size—Polystyrene particles (Bangs Labs, Fishers, Ind.) ofdifferent sizes (1, 3 and 5 micrometer diameter) were separated usingmoving optical gradient fields. Three and five micrometer diameterparticles were diluted 1/500 in distilled water and ten microliters waspipetted onto a Rain-X coated slide. The 812 system was used to generatea spot size of 25-30 micrometers consisting of 4-5 fringe periods andmoving at 15 micrometers/second.

FIG. 32 shows a sorting sequence at 1-second intervals with 3 and 5micrometer polystyrene particles. The smaller, 3 micrometer diameter,particle was readily moved by the gradient fields whereas the larger, 5micrometer diameter, particle was unaffected. The larger particle wasnot moved because it spanned multiple fringes so gradient forces wereeffectively cancelled. Similar results were obtained with 1 and 3micrometer diameter particles.

Separation By Refractive Index—Polystyrene, polymethylmethacrylate andsilica particles of similar size (˜5 micrometer diameter, Bangs Labs)and refractive indexes of 1.59, 1.49 and 1.37, respectively, were sortedby moving optical gradient fields. Observed escape velocities forpolystyrene, PMMA and silica were 44, 47 and 32 micrometers/second,respectively. Briefly, a particle is aligned in the fringe and thefringes are moved at increasing speed until the particle slips. Thisresults in a semi-quantitative measurement of the total forcesexperienced by the particle, i.e. photonic, hydrodynamic and frictional.It will be appreciated by those skilled in the art that the absolutevalue of the escape velocity will differ depending upon systemconditions, e.g., laser power. The numerical results provided herein aremeant to provide measured data for the system actually used, and are notto be considered a limitation on the values which might exist in adifferent system.

Particles were diluted 1/500 in distilled water (n=1.33). The 812 systemwas used to generate a gradient field with a fringe period of 10micrometers. Polystyrene and PMMA particles were sorted from silicaparticles by moving the gradient field at a threshold value ofapproximately 40 micrometers/second.

Separation By Surface Functionalization and Doping—Polystyrene particles(˜6 micrometer diameter) colored with blue or pink dye were purchasedfrom Polysciences, Inc. The pink particles also had carboxyl groups onthe particle surface. The particles were diluted 1/500 in distilledwater and 10 microliters was pipetted onto a Rain-X coated slide. The812 system was used to generate a moving optical gradient field with afringe period of approximately 12 micrometers. In the fringes, the pinkparticle moved preferentially.

FIG. 33 shows the actual movement of the particles.

In another experiment, 1 micrometer latex beads labeled with biotin wereused to determine changes in escape velocity when different ligands wereattached. The biotin labeled beads were diluted 1/100 in PBS buffer. A50 ul aliquot was incubated with an excess of streptavidin or 10nanometer colloidal gold-streptavidin conjugate for 10 minutes. Thebeads were pelleted by centrifugation and resuspended in PBS buffer.Measured escape velocities, using the 1064 system, were 5.3, 4.3 and 3.6micrometers/second for biotin labeled beads, beads with streptavidin andbeads with streptavidin-colloidal gold, respectively.

Separation By Wavelength Resonance (812 vs. 1064 nm)—The aboveexperiment with colored polystyrene particles was repeated using the1064 system and the results were reversed. The blue particle waspreferentially moved. Similar results were obtained when the 1064 systemwas set at 150 mW rather than 2.5 W. This suggests that wavelengthtuning could enhance the discrimination process.

Separation By Index Matching—Silica and polystyrene particles (3 and 5micrometer diameter, respectively) were diluted 1/500 in hydrophilicsilicone (dimethylsiloxane-ethylene oxide block copolymer, Gelest, Inc.,Tullytown, Pa.). The refractive index of the medium (n=1.44) wasintermediate between the silica (n=1.37) and polystyrene (n=1.59)particles. The particle size was not important in this experiment.

Using the 1064 system, the gradient force was focused into a diffusespot approx. 15 micrometers in diameter. More generally, for all of thesystems and applications described herein, a defocused beam, such as adefocused laser beam may be utilized. Preferably, the beam is defocusedsuch that the spot or beam size is on the order of magnitude of the sizeof the particle. For cells, the size would be approximately 10 to 20microns. The polystyene particle moved towards the gradient field whilethe silica particle moved away from it. This demonstrated that thesuspending medium could be changed to optimize separation.

Separation Red Blood Cells vs. Retic

A reticulocyte control (Retic-Chex) was obtained from Streck Labs. Asample containing 6% reticulocytes was stained for 15 minute s with NewMethylene Blue for 15 minutes, a nucleic acid stain that differentiallystains the reticulocytes versus the unnucleated red blood cells. Thesample was diluted 1/200 in PBS and mounted on a fluorosilane coatedslide The 812 system was used to generate optical gradient fields. Thefringe period was adjusted to 15 micrometers and was moved at 15micrometers/second. The reticulocytes were preferentially moved relativeto red blood cells.

Separation of White Blood Cells vs. Red Blood Cells

A whole blood control (Para12 Plus) was obtained from Streck Labs. Thesample was stained for 15 minutes with New Methylene Blue, a nucleicacid stain that differentially stains the nucleated white blood cellsversus the unnucleated red blood cells. The sample was diluted 1/200 inPBS and mounted on a fluorosilane coated slide. The 812 system was usedto generate optical gradient fields. The fringe period was adjusted to15 micrometers and was moved at 22 micrometers/second. The white bloodcells were moved by the fringes while the red blood cells were not.

Separation of Leukemia vs. Red Blood Cells

One milliliter of the leukemia cell line U937 suspension was pelletedand resuspended in 100 microliters PBS containing 1% BSA. Equal volumesof U937 and a 1/200 dilution of red blood cells were mixed together and10 microliters was placed on a CYTOP coated slide. Separate measurementswith moving fringe fields showed that the escape velocity for U937 cellswas sigificantly higher than the escape velocity for red blood cells, 60and 23 micrometers/second, respectively. The 1064 system was used togenerate optical gradient fields with a fringe period of approximately30 micrometers and moving at 45 micrometers/second, an intermediatefringe velocity. As expected the U937 cells move with the fringes andthe red blood cells do not. In one embodiment, the moving fringe may bereduced to a single peak. Preferably, the peak is in the form of a line.In operation, a slow sweep (i.e., at less than the escape velocity ofthe population of particles) is made across the region to beinterrogated. This causes the particles to line up. Next, the fringe ismoved quickly (ie, at a speed greater than the escape velocity of atleast some of the particle in the population), preferably in thedirection opposite the slow sweep. This causes the selective separationof those particles having a higher escape velocity from those having alower escape velocity. Optionally, the remaining line of particles maythen be again interrogated at an intermediate fringe velocity. Whilethis technique has general applicability to all of the applications andsystems described herein, it has been successfully implemented for theseparation of U937 cells from red blood cells.

Sorting of Red Blood Cells vs. Polystyrene Particles in Microchannels

Glass microchannels with an “H” configuration (see FIG. 16) were used todemonstrate sorting of red blood cells and 6 micrometer polystyreneparticles. The channels were purchased from Agilent (DNA 500 LabChip)and were 40 micrometers wide and 10 micrometers deep. Unwanted or unusedchannels and reservoir ports were blocked by backfilling with Norland 61optical adhesive followed by UV and thermal curing. The channels wereprimed with ethanol, followed by water and finally by PBS buffer with 1%BSA. The inlet reservoirs were built up about 1 mm higher than theoutlet reservoirs. Flow rates were controlled by a combination ofpressure and electrokinetic forces. A Keithley 236 power supply was usedto apply an electric field between 5 and 10 V/cm.

A 1/200 mixture of red blood cells and particles in PBS buffer, 1% BSAwas added to an inlet reservoir and an equal volume of PBS buffer, 1%BSA was added to the other inlet reservoir. The gradient field waspositioned in the crossbar of the “H” near the downstream junction. The1064 system was fitted with a cylindrical lens to increase the aspectratio of the gradient field. The resultant gradient field wasapproximately 40 micrometers wide by 80 micrometers long with a fringeperiod of 12 ums and moving at 30 micrometers/second.

In the absence of or with a nonmoving optical gradient field, the cellsand particles remain in the top half of the “H” channel and exit via theupper outlet. In the presence of a moving optical gradient field, theparticles are diverted to the lower outlet arm and are sorted from thered blood cells.

The flow rate was adjusted to approximately 80 micrometers/second. Thesorting process was digitally recorded and subsequently analyzed. Out of132 possible sorting events (121 red blood cells and 11 particles), 2red blood cells and no particles were mis-sorted. The sort rate wasapproximately 2/second.

Sorting of Red Blood Cells vs. White Blood Cells in Microchannels

FIG. 36 shows photographs of sorting of two cell types in a microchanneldevice. 1 shows a red blood cell and a white blood cell successivelyentering the moving optical gradient field. 2 shows that white bloodcell has been translated down by the action of the moving opticalgradient field while the red blood cell has escaped translation. 3 and 4show that the red blood cell and white blood cell continue to flow intoseparate channels, completing the sorting.

Gradient Force Manipulation of Liposomes

Fluorescently labeled liposomes, approximately 0.2 micrometers indiameter, were obtained from a B-D Qtest Strep kit. Ten microliters wasplaced in a Rain-X coated slide and the 1064 system was used to generatean optical gradient field. A 15 mW 532 nm diode laser was also focusedthrough the objective to visualize the liposome fluorescence. When astanding gradient field was projected onto the sample, fluorescence wasmore intense in this area. This suggests that the liposomes were movingtowards the gradient field.

Differential Motion Imaging

Polystyrene and silica particles were diluted in distilled water. Asshown in the photographs of FIG. 34, a “before” image was captured usinga CCD camera and Image Pro Express software. A moving optical gradientfield generated by the 1064 system was scanned over the particles.Another image (an “After” image) was captured and the “before” image wassubtracted. The resultant image (labeled “Difference”) clearlyidentifies that the polystyrene particle had moved.

Escape Velocities of Different Cell Types

Escape velocities were measured using a gradient field generated by the1064 system on CYTOP coated coverslips.

Cell Type Escape Velocity (um/sec.) Red Blood Cell 5.6 +/− 0.4 WhiteBlood Cell 11.0 +/− 1.8  Chicken Blood (Retic. Model) 7.3 +/− 1.4 K562Cells, No Taxol Treatment 10.0 +/− 0.7  K562 Cells, 26 Hr. TaxolTreatment 8.2 +/− 0.4 K562 Cells: Chronic myelogenous leukemia,lymphoblast

FIG. 35 shows a graph of percent of cells measured as a function ofescape velocity (μm/second).

Separation of Treated and Untreated Leukemia Cells

PMA was dissolved in ethanol at a concentration of 5 mg/mL. 3 mls ofU937 cells grown in RPMI 1640 media with supplements were removed fromthe culture flask and 1 ml was placed into each of three eppendorftubes. Cells from the first tube were pelleted for 4 minutes at 10,000rpm and resuspended in 250 uL PBS/1%BSA buffer for escape velocitymeasurements. PMA was added to the remaining two tubes of U937 cells toa final concentration of 5 ug/mL. These tubes were vortexed and placedin a 37° C. water bath for either one hour or six hours. At the end ofthe time point, the tube was removed, cells were pelleted and thenresuspended as described above and escape velocity measurements taken.The cells treated for 6 hours had a significantly higher escape velocityas compared to the untreated cells.

While preferred embodiments and methods have been shown and described,it will be apparent to one of ordinary skill in the art that numerousalterations may be made without departing from the spirit or scope ofthe invention. Therefore, the invention is not limited except inaccordance with the following claims.

We claim:
 1. A method for sorting a particle of interest from aplurality of particles comprising the steps of: determining anabsorption maxima of the particle of interest; providing a light sourceto generate a beam of coherent light at a wavelength of the absorptionmaxima; providing a plurality of particles on a support surface;illuminating the plurality of particles with a moving beam of thecoherent light, the moving beam of light causing differential movementbetween the particle of interest and the plurality of partici s; andcollecting the particle of interest.
 2. The method of claim 1, whereinthe absorption maxima is a local maxima.
 3. The method of claim 1,wherein the absorption maxima is a global maxima.
 4. The method of claim1, wherein the absorption maxima is obtained by empirical data.
 5. Themethod of claim 1, wherein the support surface is a slide.
 6. The methodof claim 1, wherein the support surface is a microfluidic channel.